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“ZOOLOGICAL
SCIENCE
An International Journal
ZOOLOGICAL SCIENCE
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ZOOLOGICAL SCIENCE 9: 679-695 (1992)
© 1992 Zoological Society of Japan
REVIEW
Hormonally Induced Synaptic Plasticity in the
Adult Neuroendocrine Brain
AKIRA MATSUMOTO
Department of Anatomy, Juntendo University School of Medicine,
Hongo, Tokyo 113, Japan
CONTENTS
MNGKOG WC HO Wee see eerie as nse nese eesaeisek tastes tetcctasiclee sires selaranistishaer sists jos aljceleisieininenintisiesinaynainslseubiepeainsleineten 679
ESHOsenicnesulatlonion synaptic plasticibyssseessesrsse-eseesare este sen dec ceceses-eeeeeercee-esecnerecesesesseceere 680
(i) Synaptic plasticity in the hypothalamus with brain lesion ...................0cceeeeceeeeecneceee eee ees 680
(ii) Synaptic plasticity in the hypothalamus without brain lesion ...................cecceeeeeeeeeeeeen ees 682
Gea) Synaptic plasticity mite VIMIN aii. fis. le osaetecGehdnceorl sane otsecnebensssentuautnssbtneeescce stencils 682
Gib) Synapticiplasticity mithe th OAG ea creeeec ee ceatein sec sensoh odes cetoecdadserdecurescdcceeceesecsecsss 683
(li-c) Synaptic plasticity during the estrous cycle ................. cee ceee cece eee eec eee eeeeeeeeeeeeseeeseeens 683
Androgenic regulation of synaptic plasticity ............. 0c. cece eee ee cece c eee ec eec eee eee eeu ereeneeeeeeeeereees 684
@ eS ynapticrplasticityaimithe:SNBccnas.cc-e heir ee pee scm once bariepct torhjans sesesepemauademeaeeceaesenciiee see 684
(ii) Plasticity of gap junctions in the SNB ............ceceeceececeeeeee eer eeneeeeteceee eee eeneeeeeeenereeeeees 685
Sex steroidal regulation of mRNA expression of structural proteins ..................cseceeeeeeeeeee eer eees 686
Gee xpressiomioige-actimvmRNA saicorecscsen crepe ceca sen eri oes sccenerinesiceessasnasherceccsnumececsranes 687
(i) aE xpressionlofocaphjunctionmmlRINAgeeescerre eee aterteeeeseesaecadeceeeseeteeeeeee cea seeee cae ceeeeae eae 688
(iii) Possible mechanisms of sex steroids for the expression of f-actin and gap junction
IRIRINIANS sc nconesaboaaaedeb soba anSaeHeodndes seGaagtsepaP ental cScosadccnedr san sense Coreanadd Seas doce nama e er eee en 689
(COMGINGION '5.cooadadbaanoasbaceran Sap ROAUHBAB ENE CHGbn AGP BASAe nace Raa tc SPeUnACHE MICA SLEn tn ioe oan ana SR ener seers aren 690
IRNGRETEN CES Reeeee ereee reset Cierny are aamrae ayers rasa tail nian’ set clase aafo sili astiais aaiisieleeiisie Muir awe tinelnadnrediata sluineina 691
neuronal number [15-17], in dendritic morphology
INTRODUCTION
It is well established that sex steroids play a
crucial role in reproductive neuroendocrine func-
tions such as gonadotropin secretion and copula-
tory behavior. The effects of gonadal steroid
hormones on the sex steroid-accumulating neuro-
nal structures have been classically subdivided into
organizational and activational ones [1-3].
Estrogen or aromatizable androgen is responsible
for modulating neuronal development and neuro-
nal circuit formation during perinatal period [4-
10]. In certain brain regions these organizational
actions of sex steroids can induce permanent sex-
ual dimorphism in nuclear volume [11-14], in
Received April 30, 1992
[18, 19], in neuronal membrane organization [20],
in the distribution patterns of serotoninergic [21]
and vasopressinergic fibers [22], in synaptic forma-
tion [23-28] and in neuronal connectivity [29, 30].
In contrast, activation effects of sex steroids on
adult brain tissues are considered to be imperma-
nent and reversible. Although the effects have been
thought to involve changes in neurophysiologi-
cal and/or neurochemical events in the neuro-
endocrine brain, evidence is now accumulating that
even impermanent effects of sex steroids can in-
volve major structural alterations in neuronal ele-
ments and circuits, reflecting several kinds of re-
productive neuroendocrine functions. Sex steroids
have been found to regulate nuclear volumen [31,
32], neuronal number [33], neuronal size [34-36],
680 A. MAtTsumMoTo
dendritic morphology [35, 37-43], vesopres-
sinergic innervation patterns [44], and synaptic
remodeling [45-51] in several areas of adult rodent
neuroendocrine brain. It has also been reported
that androgen regulates nuclear volume [52],
dendritic length [53] and synaptic reformation [54,
55] in a portion of adult canary striatum.
In the present paper, to clarify the activation
effects of sex steroids on neuronal circuit forma-
tion, the focus is placed on synaptogenic action of
sex steroids on adult neuronal structures of the
neuroendocrine brain such as hypothalamus and
spinal cord which participate in reproductive func-
tions [56-60].
ESTROGENIC REGULATION OF
SYNAPTIC PLASTICITY
The hypothalamic arcuate nucleus (ARCN),
hypothalamic ventromedial nucleus (VMN) and
preoptic area (POA), which are sex steroid-
accumulating neuronal groups [61-64] are consid-
ered to play a significant role in regulating gonado-
tropin secretion and sexual behavior [56, 57].
Most of these functions are sex steroid-dependent
and sexually dimorphic. Recently, the volume of
the POA [11-14] and VMN [12], and the synaptic
organization in the POA [23, 24], ARCN [25, 26]
and VMN [27, 28] have been reported to be
sexually dimorphic and are modified by neonatal
sex steroid environment. Estrogen during neo-
natal or prepubertal periods has a facilitatory
effect on neuritic growth of mouse hypothalamic
explants [65] or rat cultured hypothalamic cells
[66, 67], dendritic spine formation in the VMN [68]
and synaptogenesis in the ARCN [69-74], VMN
[27, 28] and POA [75]. Since these hypothalamic
neural tissues in neonatal animals are in an unde-
veloped state [28, 69, 75-80], estrogen may act as
growth-promoting factors on the developing
hypothalamic neural components and play a criti-
cal role in modulating postnatal neuronal matura-
tion and neural circuit formation in the hypo-
thalamus.
(i) Synaptic plasticity in the hypothalamus with
brain lesion
Synaptogenic action of estrogen on several neu-
ral tissues is not limited only at neonatal or pre-
pubertal period. Estrogen has been found to have
a facilitatory effect on synapse formation in the
ARCN of adult [45-49] and aged female rate [47].
Matsumoto and Arai [45-47] have pointed out
that estrogen has a facilitatory effect on synap-
togenesis in the ARCN of adult hypothalamus.
When the medial basal hypothalamus (MBH),
including the ARCN, is isolated completely by
means of Halasz knife (MBH-island), a remark-
able reduction in the number of axodendritic shaft
and spine synapses (Fig. la and b) in the ARCN of
ovariectomized females with MBH-islands is
caused by transection of the afferent fibers con-
verging to the ARCN [81]. As shown in Table 1,
the number of shaft and spine synapses decrease to
45% and 73% of the intact level, respectively.
Administration of estrogen for three weeks follow-
ing brain surgery can bring about a dramatic
increase in the number of shaft and spine synapses
in the ARCN of ovariectomized MBH-island
females. Shaft synapses restore to almost 75% of
the intact level, and the incidence of spine
synapses is significantly greater than that in control
animals.
These suggest the possibility that
estrogen can enhance the plasticity of intact arcu-
ate neurons in adult brain, presumably stimulating
not only axonal sprouting but also dendritic spine
formation in intact arcuate neurons. There are a
number of vacated synaptic sites in the ARCN
after brain surgery. Estrogen fails to increase in
the number of shaft and spine synapses in the
ARCN of ovariectomized females without brain
lesion (Table 1). This evidence indicates that
synaptogenic potential of estrogen can only be
expressed in the adult arcuate neurons when
synaptic sites are vacated due to lesion-induced
degeneration of axon terminals. In the ARCN,
most axon terminals are associated with only one
dendritic element, a shaft or a spine (Fig. la and
b). In addition to these synapses, axon terminals
contacting two postsynaptic elements in the plane
(double synapse) are seen in the ARCN (Fig. Ic).
Although the incidence of double synapses is
usually very low, three types of double synapse,
that is, spine-spine, spine-shaft and shaft-shaft
double synapses, are recognized. Treatment of
results
Synaptic Plasticity in Response to Sex Steroids 681
nbd
Poe One a
Hf
Fic. 1. (a) Axodendritic shaft synapse in
the ARCN of a female rat at two
months of age. « 33,000. (b) Axoden-
dritic spine synapse in the ARCN of a
female rat at two months of age. S=
spine. 28,000. (c) Spine-spine dou-
ble synapse in the ARCN of an ovar-
iectomized MBH-island female rat
treated with estrogen for three weeks.
S=spine. 24,000. (Reproduced
with permission from Ref. [46])
TABLE 1. Effect of estrogen on numbers of axodendritic shaft and spine synapses in the
arcuate nucleus (ARCN) with or without complete deafferentation of the medial
basal hypothalamus (Reproduced with permission from Ref. [46])
Axodendritic synapse
Group ae ;
Shaft synapse Spine synapse
Intact controls 8 1544+91* 215+23
OVX 6 1539 + 87 207 +23
OVX+EB 6 1686 + 153 241 +37
MBH-island+ OVX 9 696+ 46 156+ 20
MBH-island+OVX-+ EB 8 1159+70 380+ 24
* Mean+S.E.M.
MBH-island=complete deafferentation of the medial basal hypothalamus; OVX=ovariec-
tomy; EB=treatment of estradiol benzoate.
682 A. Matsumoto
TABLE 2. Effect of estrogen on numbers of double synapses in the arcuate nucleus (ARCN) with or
without complete deafferentation of the medial basal hypothalamus (Reproduced with permission
from Ref. [46])
Number Double synapse
Guo of rats .
Spine-spine Spine-shaft Shaft-shaft
Intact controls 8 1.87+0.58* 3.75 +1.01 4.75+1.47
OVX 6 1.66+0.67 2.83+0.79 3.00 + 1.00
OVX+EB 6 2.83 £0.94 7-33 ste1e45 5.00+0.51
MBH-island+OVX 9 1.44+0.50 6.77 + 1.43 4.11+1.12
MBH-island+OVX+EB 8 15.00 +3.45 ES ae silk 6.00 + 1.03
* Mean+S.E.M.
MBH-island=complete deafferentation of the medial basal hypothalamus, OVX=ovariectomy, EB=
treatment with estradiol benzoate.
estrogen causes a dramatic increase in the numbers
of spine-spine and spine-shaft double synapses in
the ARCN of ovariectomized MBH-island rats
(Table 2). Although the total number of double
synapses is very low as compared to regular types
of synapses, an increase in the number of spine-
spine and spine-shaft double synapses, but not of
shaft-shaft double synapses, is remarkable in the
deafferented ARCN of estrogen-treated females
(Talbe 2). The findings provide further support for
the ability of estrogen to stimulate synaptogenesis
in the adlut ARCN. Collateral sproutings from the
axon terminal, which have already synapsed on a
shaft or a spine may not only reoccupy a vacated
shaft or spine, but may also synapse with a spine
newly formed by estrogen stimulation. Much
remains to be clarified concerning the functional
significance of this plastic response of adult neural
tissues to estrogen. It seems unlikely that synaptic
connections established at early developmental
period persist throughout the life. In this context,
it is worthy to note the coexistence of growth cones
and degenerating axon terminals in the adult VMN
[82], indicating that spontaneous synaptic degen-
eration and synaptic remodeling can occur in adult
brain. Estrogen might be responsible for such a
process of synaptic reorganization.
(ii)
out brain lesion
Synaptic plasticity in the hypothalamus with-
Estrogen has been reported to exert a stimula-
tory influence on synaptogenesis in the VMN [51,
83], in the lateral septum [84] and in the midbrain
.
central gray [85] of adult female rats even if the
neural tissues are never lesioned.
(ii-a) Synaptic plasticity in the VMN
The VMN plays a crucial role in female sexual
bahavior or lordosis, which is activated by estrogen
and progesterone during the estrous cycle [57, 58].
Since the VMN contains a number of estrogen-
accumulating neurons [61-64], and implants of
estrogen into the VMN facilitate lordosis [58], the
VMN is thought to be a direct site for estrogenic
effect on lordosis. Administration of estrogen into
ovariectomized female hamsters for two days in-
duces a 48% increase in the dendritic length of
VMN neurons compared with VMN neurons of
control females [42]. The formation of dendritic
spines on VMN neurons of ovariectomized female
rats is also stimulated by estrogen treatment for
two days [39, 40]. This estrogenic regulation of
dendritic membrane area implies a concomitant
change in synaptic inputs to dendrites. Ultra-
structural study reveals estrogenic influence on
synaptic inputs to VMN neurons [51, 83]. Accord-
ing to Frankfurt and McEwen [51], axodendritic
synapses cover 81% of the dendritic membrane in
ovariectomized rats treated with estrogen for two
days, and 53% in ovariectomized ones. The num-
ber of axodendritic synapses per unit area of the
VMN is 2.93+0.13 in ovariectomized rats treated
with estrogen, and 1.79+0.11 in ovariectomized
ones. Changes in the dendritic length, dendritic
spine density and axodendritic synaptic population
in the VMN have been detected as early as two
Synaptic Plasticity in Response to Sex Steroids 683
days after estrogen treatment. These morpholo-
gical findings suggest that the VNM might be
responsive to even daily changes in estrogen level.
(ii-b) Synaptic plasticity in the POA
The luteinizing hormone-releasing hormone
(LHRH) neurons in the POA form the final com-
mon pathway regulating the gonadotropin secre-
tion from the anterior pituitary [86]. The activity
of LHRH neurons is considered to be regulated by
intra- and extrahypothalamic neural elements as
well as steroidal environment [56, 57]. Since
LHRH neurons do not accumulate estrogen [87], it
seems likely that other estrogen-sensitive inter-
neurons in the POA transduce steroidal signals
and in turn, alter the activity of LHRH neurons.
In fact, LHRH-immunoreactive terminals have
been reported to make synaptic contact with
estrogen receptor-immunoreactive neurons in the
POA [88]. Immunohistochemical ultrastructural
investigations have demonstrated that LHRH
neurons themselves in the POA are contacted with
axon terminals containing amino acid, mono-
amines and neuropeptides such as y-aminobutyric
acid [89, 90], dopamine and norepinephrine [91,
92], serotonin [94], @-endorphin [92, 93], subst-
ance P [95], pro-opiomelanocortin [96], adreno-
corticotropic hormone [97] corticotropin-releasing
factor [98], and LHRH [99-102]. According to
Chen et al. [93], there is sexual dimorphism in the
synaptic input to LHRH neurons in the rat POA.
LHRH neurons in females receives approximately
twice the number of synapses as do those of males.
f-endorphin-immunoreactive terminals contribute
to this dimorphism. Physiological differences in
regulation of gonadotropin secretion may be
reflected in a sexual dimorphic connectivity of the
LHRH system.
Witkin et al. [102] have demonstrated that
synaptic input and glial apposition to LHRH
neurons in the POA of adult rhesus monkeys is
sensitive to circulating estrogen. Removal of
estrogen by ovariectomy results in a significant
increase in the apposition of glial processes to
LHRH-immunoreacive neurons and a significant
decrease in their innervation in the POA, and
these changes are prevented by estrogen replace-
ment. This anatomical study indicates the possibil-
ity that LHRH system responds to alterations in
the gonadal steroid environemtn with reversible
changes in synaptic input and glial ensheathing. In
relation to this finding, it is noteworthy that so-
mata of LHRH-immunoreactive neurons in the
POA of anestrous sheep are less innervated than
non-immunoreactive cells around LHRH-im-
munoreactive neurons, and that somata and de-
ndrites of LHRH-immunoreactive neurons are
almost entirely surrounded by glial processes
[103]. Thus, natural seasonal changes in estrogen
level probably influence the synaptic organization
of LHRH system.
(ii-c) Synaptic changes during the estrous cycle
As mentioned above, estrogenic effects on
changes in neuronal elements in the VMN occur
two days after estrogen treatment. It seems quite
possible that daily fluctuations in the level of
estrogen during the estrous cycle cause significant
alterations in the amount of membrane available
for synaptic input and in the number of synapses.
The dendritic spine density of VMN neurons of
female rats at proestrus has been reported to be
significant higher than that at estrus and diestrus
[39]. It has not yet determined whether these
changes in dendritic spines reflect changes in
synaptic population in the VMN during the estrous
cycle. On the other hand, Olmos et al. [104] found
that the number of axosomatic synapses in the
ARCN decreases at estrus when compared to
other days of the estrous cycle. There is a reduc-
tion of 31% in axosomatic synapses between
proestrus and estrus. Because estrogen has a
facilitatory effect on synaptic formation in the
adult ARCN, it is reasonable to assume that
synaptic remodeling of the ARCN occur during
the estrous cycle. It seems likely, therefore, that
the neuronal plasticity which has been demon-
strated to occur during the estrous cycle may be a
natural consequence of sex steroid feedback, and
that the adult pattern of synaptic connections can
be altered in the hypothalamus in relation to
reproductive neuroendocrine and behavioral func-
tions.
Although the hippocampus is not considered to
be directly involved in regulatory mechanisms of
neuroendocrine functions, estrogen exerts a stim-
684 A. Matsumoto
ulatory influence on dendritic spine formation of
pyramidal neurons in the hippocampus (CAI re-
gion) of ovariectomized adult rats [105]. More-
over, spine density of the CA1 pyramidal neurons
[106] and synaptic population in the neuropil of
CA1 region alter during the estrous cycle
(McEwen, B. S., personal communication). These
evidence further supports the possibility that
neuronal elements and synaptic connections
reorganize during the estrous cycle.
ANDROGENIC REGULATION OF
SYNAPTIC PLASTICITY
The spinal nucleus of the bulbocavernosus
(SNB) of the rat lumbar spinal cord is a sexually
dimorphic motoneuron pool [107-111]. The SNB
motoneurons innervate the perineal striated mus-
cles bulbocavernosus and levator ani that attach to
the penis. They have an important role in copula-
tory behavior, which is sensitive to alterations in
circulating levels of androgen in adulthood [59,
60]. Both neuronal and muscular elements contain
androgen receptors [107-114]. Castration of adult
male rats rsults in a significant decrease in both
somatic size [34, 36] and dendritic length [37, 38,
41] of these motoneurons, and androgen treatment
of castrates reverses this effect. According to Kurz
et al. [43], androgenic influences on somatic size
and dendritic length are also detected in another
androgen-sensitive motoneuron pool in the lumbar
spinal cord, the dorsolateral nucleus (DLN), which
innervates the ischiocavernosus and urethral
sphincter muscles [113, 115]. These evidence
suggests that the adult SNB and DLN
motoneurons retain a great deal of plasticity to
androgen, which is expressed when androgen
levels fluctuate. This androgenic regulation of
somatic and dendritic membrane area implies a
Fic. 2. Percentage of SNB membrane covered by
synaptic contacts (a), size of synaptic contacts (b)
and frequency of synaptic contacts per 1 ~m of SNB
neuronal membrane (c) of control (Controls), cas-
trated (Castrates) and castrated male rats treated
with testosterone for four weeks (Castrates + Testos-
terone). Vertical lines indicate S.E.M. Numbers in
the bottom of columns refer to the number of rats
examined. (Reproduced with permission from Ref.
[2])
concomitant change in synaptic inputs to these
membranes.
(i) Synaptic plasticity in the SNB
Quantitative electron microscopic study reveals
a | Soma
| | Proximal dendrite
30+
155
5 5 5
Controls Castrates Castrates
Percent Covering of Synaptic Contacts
+
Testosterone
b EI Soma
HEE Proximal dendrite
(um)
2.05
io
iS)
2
r=
cS)
ra 9]
a
°
c
bal
a
r)
ao
pe
7)
5 5 5
Controls Castrates Castrates
+
Testosterone
Cc =] Soma
HEE Proxima! denarite
9
ty
a
(o}
r
0.100-
Frequency of Synaptic Contacts
(per jum of Neuronal Membrane)
5 5 5
Costrates
Controls Castrates
+
Testosterone
Synaptic Plasticity in Response to Sex Steroids 685
androgenic influences on synaptic inputs to SNB
motoneurons [48, 50]. In intact adult males,
synaptic structures cover about 40% and 47% of
somatic and proximal dendritic membranes of
SNB motoneurons, respectively (Fig. 2a) [48].
Four weeks after castration, these values are re-
duced to about 14% and 19% of somatic and
proximal dendritic membranes, respectively.
However, treatment of testosterone for four weeks
after castration prevents this decline. Castration
and testosterone treatment also influence the
synaptic size (Fig. 2b) and number of synaptic
contacts per unit length of somatic and proximal
dendritic membranes (Fig. 2c). These results indi-
cate that androgen is critical for maintaining the
reformation of synaptic inputs to SNB
motoneurons in adult male rats. Together, the
earlier light microscopic studies [34, 36-38, 41]
and ultrastructural ones suggest that the SNB
system can retain a great deal of plasticity in
adulthood. Because androgen levels in rodents
fluctuate with seasons [38], stress and aging [116,
117], the synaptic changes induced by castration
may reflect an ongoing process of synaptic or-
ganization and reorganization regulated by
androgen throughout the adult life.
Previous studies have suggested which kinds of
neurotransmitters or neuromodulators may be
contained in affectes to SNB motoneurons. Im-
munohistochemical studies have suggested that the
presence of norepinephrine, serotonin, substance
P, leu- and met-enkephalin, thyrotropin-releasing
hormone (TRH) and calcitonin gene-related pep-
tide (CGRP) in fibers and terminals in close prox-
imity to somata and dendrites of motoneurons in
the rat SNB [118, 119], and in the cat or baboon
Onuf’s nucleus which is homologous to the SNB
[120-124]. These data indicate that the terminals
of these fibers may make synaptic contact with
SNB neurons. In fact, serotonin- and tyrosine
hydroxylase-immunoreactive terminals make
synaptic connection with SNB motoneurons [125].
Motoneurons in the Onuf’s nucleus are contacted
with axon terminals containing norepinephrine,
serotonin, substance P, leu- and met-enkephalin,
TRH and CGRP [120, 124]. It has been clearly
established that synaptic inputs to the SNB
motoneurons can be regulated by androgenic in-
fluence. Further studies are needed to determine
the chemical identity of neuronal inputs to the
SNB motoneurons which are affected by
androgen.
(ii) Plasticity of gap junctions in the SNB
The SNB motoneurons exist in a cluster, and
their somata come into contact with each other
[107-111]. Numerous number of longitudinally
oriented bundles of dendrites are packed in the
neuropil of the SNB [126, 127]. Gap junctional
plaques are found between these motoneurons,
Fic. 3. A gap junction (arrow and inset) can be seen
along the apposed membranes of a proximal den-
drite (PD) of an SNB motoneuron and another SNB
dendrite (D) in a testosterone-treated male rat four
weeks after castration. Cholera toxin conjugated to
HRP (CT-HRP) was injected into the bulbo-
cavernosus muscles two day before autopsy, and the
spinal cords containing SNB were dissected, proces-
sed with a modified tetramethylbenzidine method
for visualization of retrogradely transported CT-
HRP. CT-HRP crystals (arrowheads) are found in
both dendrites. x 16,400 (inset: x 175,500). (Re-
produced with permission from Ref. [128])
686 A. Matsumoto
TABLE 3.
Effect of androgen on the incidence and size of gap junctions between SNB motoneurons in
control and experimental animals (Reproduced with permission from Ref. [128])
Number of neurons
Number of gap
Number - : : Length of gap
containing ga t : ;
Group of rats Finclone P ai junctions (nm)
Controls 5 2.6+0.4* 3.4+0.4 168.8+25.2
Castrates 0.8+0.2' On On 64.74 8.1
Castrates + testosterone 2.8+0.5 3.6+0.2 154.4+19.1
* Mean+S.E.M.
* P<0.001, * P<0.0005 (vs. Controls or Castrates+ testosterone).
using thin-section and freeze-fracure technique
[128]. The presence of gap junctions between SNB
motoneurons is also ientified with electrophysiolo-
gical and Lucifer yellow dye coupling techniques
[129]. Translucent electron microscopic study re-
veals that gap junctions show a characteristic fea-
ture of pentalaminar structure (Fig. 3). Freeze-
fracture replicas demonstrate plaques of in-
tramembranous particles of the protoplasmic and
complementary pits on the extracellular fracture
faces. These particles are considered to be chan-
nels of gap junctions, permitting electrical and/or
metabolic coupling between motoneurons. Of the
gap junctions observed in the SNB, 45% were
somatodendritic, 35% dendro-dendritic and 20%
somatosomatic. As shown in Table 3, the removal
of testosterone by castration dramatically reduces
the number and size of the junctional plaques, and
these changes are prevented by testosterone re-
placement. Because the size of the junctional
plaques is related to the number of gap junctional
channels between the cells [130], the findings that
androgen regulates the total number of gap junc-
tional channels would indicate that the degree of
electrical and/or metabolic coupling is regulated
by androgen. The enhanced electrical and/or
metabolic coupling between SNB motoneurons
may promote synchronous neuronal activity in this
nucleus. Gap junctional plaques are also found
between motoneurons in the DLN [131].
In addition to SNB motoneurons, gonadal ster-
oids have been reported to regulate the size or
frequency of gap junctional plaques in various
kinds of cells. For example, estrogen increases gap
junctions between granulosa cells of the rat ovary
[132, 133], between smooth muscle cells of rodent
uterus [134-139] and between neurons in the rat
ARCN [140]. In contrast, removal of estrogen by
ovariectomy induces an increase in the incidence
of Lucifer yellow dye coupling between mag-
nocellular neurons in the supraptic nucleus of the
rat hypothalamus [141]. On the other hand,
androgen stimulates the incidence of Lucifer yel-
low dye coupling between magnocellular neurons
in the paraventricular nucleus of the rat hypothala-
mus [142]. Female Xenopus frogs possess dye-
coupled laryngeal muscle fibers, whereas adult
males do not [143]. Androgen treatment of
females reduces the coupling in females. These
evidence indicates that gonadal steroids regulates
the number of gap junction channels in a wide
variety of reproductive tissues, including the
gonads, nervous system, and smooth and striated
muscles. These tissues have in common the pre-
sence of gonadal hormone receptors. Indeed, it
become reasonable to ask whether steroid control
of gap junctions and electrical coupling is a com-
mon feature of all such steroid-sensitive cells.
SEX STEROIDAL REGULATION OF
mRNA EXPRESSION OF STRUCTURAL
PROTEINS
Although the molecular mechanisms underlying
the neuronal plasticity in response to sex steroids
are poorly understood, evidence is now accumulat-
ing that sex-steroids play a significant role in
regulating gene expression of structural proteins in
sex steroid-sesitive neuronal tissues [144-149].
Synaptic Plasticity in Response to Sex Steroids 687
Riri ae of
Bee
bea) &
eget teeta ra ;
pate soir ea eH eRe
ar
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Ea a Aghines ones Eng re Boies se iat,
ian z ea iets
Fic. 4. Autoradiographic signals of £-actin mRNA on SNB motoneurons in a castrated male rat treated with
testosterone for four weeks (a), and in a castrated male rat (b). X390. (Reproduced with permission from Ref.
[149])
(i) Expression of B-actin mRNA
Actin is one of the important cytoskeletal pro-
teins that has been identified in neurons of the
central nervous system [150-152]. Actin filaments
are found to associate particularly with the neuro-
nal membrane and postsynaptic density. As men-
tioned before, androgen regulates the somatic and
dendritic membrane area of SNB motoneurons
and the number of synapses onto these
motoneurons. It seems likely, therefore, that actin
might be involved in the hormonally induced
neuronal plasticity in the SNB motoneurons.
In situ hybridization histochemical study using
complementary DNA encoding chick /-actin re-
veals androgenic influences on the expression of
f-actin mRNA in SNB motoneurons of adult male
rats [149]. Hybridizable G-actin mRNA are found
to localized in the somata and proximal dendrites
of SNB motoneurons (Fig. 4a and b). As shown in
Figs. Sa, 5b and 5c, removal of androgen by castra-
tion significantly reduces the neuronal area of SNB
motoneurons and the expression level of -actin
mRNA in SNB motoneurons, whereas these
changes are prevented by testosterone treatment
for four weeks. On the contrary, castration or
testosterone treatment does not induce any change
in the expression level of ~-actin mRNA in the
motoneurons of the retrodorsolateral nucleus
(RDLN) (Fig. 5a-c). The RDLN motoneurons
innervating hindlimb musculature are examined,
because the RDLN motoneurons are known to be
androgen-insesitive [148, 149, 153]. These findings
suggest that androgen exerts a regulatory influence
on the f-actin mRNA expression in the SNB
motoneurons. {-tubulin is known to be main
component of microtubules which are another
major cytoskeletal element in neuronal cells. The
expression of #-tubulin mRNA in the SNB moto-
nourons is also regulated by androgen (Matsumo-
to, A. et al., unpublished data).
Recent studies provide evidence for an increase
in expression of cytoskeletal proteins such as actin,
tubulin, and their mRNAs in neural plasticity
processes like neuronal growth and synapse forma-
tion [154-158]. In the SNB, androgen has been
shown to regulate the somatic and dendritic size of
motoneurons [34, 36-38, 41] and the number and
size of synapses onto these motoneurons [48, 50],
suggesting that f-actin participates in the under-
lying mechanisms of plastic response of SNB
motoneurons. Estrogen has been reported to exert
a stimulatory influence on the expression of actin
[159] and f£-actin mRNA in immature rat uterus
[160]. These findings indicate the possibility that
sex steroids generally play a significant role in the
expression of G-actin genes in sex steroid-sensitive
cells.
688 A. Matsumoto
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Fic. 5. Neuronal area (a) and numbers of grains of
B-actin mRNA per neuronal area (b) and per unit
area (c) of motoneurons in the SNB and RDLN.
Solid bars, control males; hatched bars, castrated
males; open bars, castrated males treated with tes-
tosterone. Six animals were examined in each
group. Values given are the mean+S.E.M. (Repro-
duced with permission from Ref. [149])
(11) Expression of gap junction mRNA
Recent biochemical and molecular biological
studies make possible to raise polyclonal and
monoclonal antisera against several kinds of gap
junction proteins (connexins) and to clone cDNAs
coding for them [161-163]. It has been reported
that neuronal tissues react immunohistochemically
with antibody against gap junction protein of the
rat liver (connexin 32) [164-169], and that the
expression of gap junction mRNA is demonstrated
in the neural tissues by a Northern blot analysis
[170, 171] and by in situ hybridization histochemis-
try using complementary DNA (cDNA) encoding
connexin 32 [148, 149, 169, 172].
Androgenic influences on the expression of gap
junction mRNA in the SNB motoneurons have
been clarified by recent in sity hybridization his-
tochemical studies using cDNA encoding connexin
32 [148, 149]. Hybridizable gap junction mRNA
are localized on the somata and proximal dendrites
of SNB motoneurons (Fig. 6a and b) [148]. Re-
moval of androgen by castration significantly re-
duces the expression level of gap junction mRNA
in the SNB motoneurons, whereas this decline is
prevented by testosterone treatment for two days
(Fig. 7a-c). The recovered expression level is
almost the same as that treated with testosterone
for four weeks [149]. This evidence indicates that
androgen can enhance and maintain the expression
level of gap junction mRNA in the SNB
motoneurons. On the contrary, castration or tes-
tosterone replacement does not induce any change
in the expression level of gap junction mRNA in
the motoneurons of the RDLN (Fig. 7a-c). These
results indicate that androgenic regulation of gap
junction gene expression is rather specific to the
SNB.
The recovery of expression level of the gap
junction mRNA is detected in the SNB
motoneurons of castrates treated with testoster-
one. This evevation of gap junction transcript is
thought to be associated with the synthesis of gap
junction protein, reflecting the formation of gap
junction channels and its plaques. Indeed, we
have recently pointed out that androgen increases
the number and size of junctional plaques between
SNB motoneurons [128]. In the uterine myomet-
rial cells, an increase in the number and size of gap
junctional plaques has been observed at parturi-
tion [173]. Plasma levels of estrogen [174] and
number of estrogen receptors in these cells [175]
increase markedly, preceding the increment of gap
junctions observed at parturition. Formation of
gap junction plaques between these cells is en-
hanced by treatment with estrogen [134-139]. In
addition to these obervations, as increase in the
expression of gap junction mRNA had been de-
tected in these cells at parturition [176]. These
Synaptic Plasticity in Response to Sex Steroids 689
ees Baas ce ees RAYS et at
Ro Se ewhaces S — eh".
Fic. 6. Autoradiographic signals of gap junction mRNA on SNB motoneurons in a castrated male rat treated with
testosterone for two days (a), and in a castrated male rat (b). X390. (Reproduced with permission from Ref.
[148])
Neuronal area( x10 °mm’)
Number of grains/Neuronal area
500
250
”
Z
00)
D
S)
[—
Z
SNB RDLN
Number of grains/Unit area
Lia Ya
SNB RDLN
Fic. 7. Neuronal area (a) and numbers of grains of gap
junction mRNA per neuronal area (b) and per unit
area (c) of motoneurons in the SNB and RDLN.
Solid bars, control males; hatched bars, castrated
males; open bars, castrated males treated with tes-
tosterone for two dyas. Six animals were examined
in each group. Values given are the mean+S.E.M.
(Reproduced with permission from Ref. [148])
findings indicate that sex steroids generally exert
influences on regulating the expression of gap
junction genes in sex steroid-sensitive cells.
(iii) Possible mechanisms of sex steroids for the
expression of B-actin and gap junction mRNAs
As mentioned above, removal of androgen by
690 A. MATSUMOTO
castration reduces the expression of f-actin and
gap junction mRNAs in SNB motoneurons and
this decline is prevented by testosterone treat-
ment. In contrast to the SNB motoneurons,
androgen does not induce any change in the ex-
pression of @-actin and gap junction rRNAs in the
RDLN motoneurons. These evidence indicate
there is regional difference in regulatory mecha-
nisms of androgen on the ejxpression of /-actin
and gap junction mRNAs in these lumber moto-
neurons.
According to Breedlove and Arnold [107, 113]
and Jordan et al. [114], there is a regional differ-
ence in the pattern of androgen accumulation by
lumbar motoneurons, that is, the motoneurons in
the SNB accumulate androgen more frequently
and densely than those in the RDLN. This evi-
dence may be correlated with the difference in
androgenic influence on the expression of /-actin
and gap junction mRNAs between these two
motoneuron pools. It seems likely, therefore, that
androgenic regulation of the expression of these
genes is specific to the SNB, and that the express-
ion of #-actin and gap junction genes in androgen-
accumulating neurons is sensitive to androgen.
The mechanism by which sex steroids influence
on the expression of f-actin and gap junction genes
is not yet known. The observed modulation of
their mRNA expression may be attributed to al-
teration in transcription rate of genes or in stability
of mRNAs [177]. Steroid hormones have been
shown to regulate the gene expression first by
binding to their specific receptors and then by
interaction of the hormone-receptor complex with
specific steroid hormone responsive elements on
the DNA which act as transrcriptional enhancers
[178-181]. To clarify the regulatory mechanisms
of the #-actin and gap junction gene expression, it
will be important to identify the steroid hormone
responsive elements in these genes. However, the
steroid hormone responsive elements have not yet
been detected in the rat Z-actin gene [182]. It is
possible that there are no steroid hormone respon-
sive elements in these genes. The conjecture may
be associated with the idea that expression of these
genes is mediated by some gene products which
are under the direct influence of the androgen-
receptor complex.
There is another possible explanation for the
regional difference in androgenic regulation of the
expression of #-actin and gap junction mRNAs
between SNB and RDLN motoneurons. It has
been suggested that the androgenic regulation of
the size of SNB motoneurons is mediated through
their target muscles [183]. According to Araki et
al. [184], androgen does not induce any change in
neuronal size of the SNB motoneurons which have
innervated androgen-insensitive soleus muscle. In
contrast, the size of SNB motoneurons which have
reinnervated to their androgen-sensitive target
muscles is reduced by castration and enlarged by
testosterone treatment. Recently, it is reported
that androgen is involved in modulating the ex-
pression of calcitonine gene-related peptide
mRNA in SNB motoneurons through the effects
on their target muscles [185]. On the other hand,
in the RDLN system, Leslie et al. [153] pointed out
that castration has no effect on the size of RDLN
motoneurons and their target muscle, flexor digi-
torum brevis. These findings indicate the possibil-
ity that, in the SNB system, androgen plays a
significant role in the production of muscle-derived
trophic factors which can regulate the size of SNB
motoneurons. It seems likely, therefore, that
androgen regulates not only the neuronal size of
SNB motoneurons but also the expression of /-
actin and gap junction genes in them through its
action on their target muscles.
CONCLUSION
The present paper provides ultrastructural evi-
dence suggesting that sex steroids play a significant
role in modulating neural circuit formation in sex
steroid-sensitive areas in adult neuroendocrine
brain. Electrical coupling and/or cell-to-cell com-
munication via gap junctions may also be modified
by sex steroid environment. Moreover, we now
undertake to clarity the molecular mechanisms
underlying the neurnal plasticity in response to sex
steroids. Androgen exerts potent influences on
modulating gene expression of structural proteins
such as #-actin, 8-tubulin and gap junction in the
SNB motoneurons. Expression of growth-
associated protein 43 kDa (GAP-43) mRNA in the
VMN is reported to be regulated by estrogen
Synaptic Plasticity in Response to Sex Steroids 691
[147]. In relevant to these evidence, it is of interest
to note that the expression of actin and tubulin
mRNAs is facilitated by neonatal treatment with
androgen in the preptic area-hypothalamus [145]
and limbic system of female rats [144]. The
expression pattern of @-tubulin mRNA in the
developing rat hypothalamus is sexually dimorphic
[146]. Exposure to androgen dring neonatal
period has a permanent organizing effect on
neuronal morphology and synaptic pattern in these
regions [1-10] which contain abundant receptors
for sex steroids [186, 187]. Thus, sex steroids may
be involved in neuronal plasticity, that is, organi-
zation and reorganizatin of neuronal elements and
synaptic connections, through regulation of gene
expression for neuronal structural proteins such as
actin, tubulin, GAP-43 and gap junction protein.
ACKNOWLEDGMENTS
The present author’s studies were carried out in col-
laboration with Prof. Y. Arai and Mrs. S. Murakami,
Department of Anatomy, Juntendo University School of
Medicine, Tokyo, Prof. A. Urano, Ocean Research
Institute, University of Tokyo, Tokyo, Dr. S. Hyodo,
Department of Biology, College of Arts and Sciences,
University of Tokyo, Tokyo, and Profs. A. P. Arnold
and P. E. Micevych, Laboratory of Neuroendocrinology,
Brain Research Institute, University of California at Los
Angeles, Los Angeles. These studies were supported by
the grants from Ministry of Education, Culture and
Science of Japan, Narishige Neuroscience Foundation,
Yamada Science Foundation and NIH (NS23468 and
HD15021).
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ZOOLOGICAL SCIENCE 9: 697-712 (1992)
REVIEW
The Species Concept in Theory and Practice
WALTER J. Bock
Department of Biological Sciences, Columbia University,
New York NY 10027, U.S.A.
ABSTRACT—The species concept has always been an integral part of basic biological theory and is
not primarily a part of systematics. The biological species concept is based on evolutionary theory and
applies only to sexually reproducing organisms. The important aspect of the species concept is lack of
gene flow between different species, and hence the defining criterion of the species is genetic isolation.
Species possess three different major properties, namely genetic isolation, reproductive isolation and
ecological isolation; these properties evolve at different times and under the effect of different causes
during the speciation process. Speciation requires an external isolating (geographic-ecological) barrier
during the initial allopatric phase in which period genetic isolation evolves and must reach 100%
efficiency. The subsequent sympatric phase of speciation occurs after the disappearance of the
external isolating barrier when members of the two newly evolved species can interact with one
another and exert mutual selective demands on one another. Much of the reproductive and ecological
isolation evolves during this secondary sympatric phase. The practical application of the species
concept in systematics and the recognition of species taxa requires many arbitrary decisions. No single
Species notion exists which can be applied to all organisms, sexually and asexually reproducing.
Recognition of species taxa in asexually reproducing organisms is based on amount of variation and
gaps in the variation of phenotypic features associated with ecological attributes of these organisms as
compared to similar attributes in sympatric species taxa of sexually reproducing organisms. Because
recognition of species taxa, including those in sexually reproducing organisms, depends on many
arbitrary decisions especially when dealing with broad geographical and temporal ranges, species taxa
in general cannot be used as the foundation for developing and testing theoretical concepts in
evolutionary theory. This can only be done with the non-dimensional species concept and with species
taxa that match this concept closely.
© 1992 Zoological Society of Japan
INTRODUCTION
Perhaps more problems exist in understanding
the species concept than any other basic theoreti-
cal idea in biology in spite of excellent analyses of
this concept by Mayr [1-9] and others. Much of
this confusion apparently arises from the
erroneous belief accepted by most workers that the
theoretical species concept lies within the purview
of systematics and the resulting attempt to develop
a single species concept applicable to all organisms
[10]. This is simply not possible. Rather, the
species concept is part of basic biological theory
and as such is applied as closely as possible to
Received May 29, 1992
objects in nature (=species taxa) by systematic
biologisis. Moreover, the species concept has
changed fundamentally over the decades and with
these changes, our ideas on species taxa have
modified accordingly. From the earliest days of
biological thinking until sometime after Darwin
published his “On the origin of species”, the
species concept was based on typological essential-
ism following the ideas of Plato and other early
philosophers. The typological species concepts is
usually, but erroneously, called the morphological
species concept and was applied equally to all
organisms, both those reproducing sexually and
asexually. Species taxa were recognized morpho-
logically. Geographic variants were treated as
different species, as were many distinctive morphs
698 W. J. Bock
within populations. As evolutionary ideas became
better known, the basis of the species concept
modified gradually to that of evolutionary theory.
By the early years of the 1940’s many, but not all,
workers based the species concept firmly on evolu-
tionary theory with the biological species concept
being the generally accepted one following Mayr
[1, 2]. The biological species concept applies only
to sexually reproducing organisms, but most syste-
matists do not make this distinction when describ-
ing species taxa which are still recognized morpho-
logically. However, morphs within populations,
when recognized, are no longer considered as
species and geographic variants are grouped
together into broadly based polytypic species taxa.
Yet many workers continue to treat the species
concept as if it was part of systematics and to
believe that a single species concept exists and can
be applied to all organisms, sexually and asexually
reproducing. Although earlier comments may
exist, the first clear statement that the species
concept is not a part of systematics, but rather is an
integral part of evolutionary theory, was by Szalay
and Bock [11].
This review of the species concept will be found-
ed on three fundamental points, namely: (a) The
theoretical species concept (the non-dimensional
species concept) is part of evolutionary theory and
applies only to sexually reproducing organisms.
(b) Recognition of species taxa in nature is part of
systematics practice and for sexually reproducing
organisms (the multi-dimensional species notion),
species taxa should be as close to the theoretical
concept as possible. (c) No single species idea
exists which can be applied equally well to all
organisms.
SPECIES—THE THEORETICAL DEFINITION
A) Species versus phyletic lineages
If the species concept is based on evolutionary
theory, the first decision is which unit within this
theory is to be considered the species. This
problem arises because two distinctly different but
related units have been labelled as the species by
different workers. These are: (1) the complex of
interbreeding individual organisms co-existing at
one point in time which are genetically isolated
from other such complexes; and (2) the time-line
of this complex of interbreeding organisms result-
ing from it reproducting itself generation after
generation. Only the first is properly termed the
species; the second is the phyletic lineage [12, 13].
Several species concepts—the evolutionary, the
phylogenetic and the cladistic—are actually seg-
ments of phyletic lineages. One must be most
careful in reading the biological and philosophical
literature to determine whether the author actually
means “the species” or really means “the phyletic
lineage” when the term species is used; often it is
not possible to determine which of these two units
was means and the real meaning of the analysis
cannot be ascertained.
B) The nondimensional species concept
In the decades following Darwin, biologists
struggled to ascertain the core of the species
concept and finally agreed that species are groups
of interbreeding individuals which have vertical
flow of genetic material during the production of
offspring, and that the boundary between species
is genetic isolation or the lack of vertical gene flow
between members of different species. That is,
members of a species can interbreed with one
another and contribute genes to the production of
offspring, but members of different species cannot
do so. Genetic isolation became accepted during
the evolutionary synthesis as the criterion for the
species concept. Vertical gene flow bewteen spe-
cies is achieved only by interbreeding (i.e., repro-
duction) between them. But evolutionists con-
fused genetic isolation with repreductive isolation.
And hence the species concept was defined in the
period of the evolutionary synthesis as “Species are
groups of actually or potentially interbreeding natu-
ral populations, which are reproductively isolated
from other such groups” [2]. Unfortunately, evo-
lutionists failed to recognize the asymmetry be-
tween reproductive isolation and genetic isolation
between speies. Species which are reproductively
isolated are genetically isolated but species which
are genetically isolated may not be reproductively
isolated. The horse and donkey represent two
species which are genetically isolated but not re-
productively isolated, at least under captive condi-
The Species Concept 699
tions if not in the wild. This confusion between
genetic and reproductive isolation resulted in dis-
agreements on aspects of speciation such as
whether selective demands can increase genetic
isolation from less than 100% to 100% after sym-
patry has been established between newly evolved
species (see Koopman [14] for an example of this
problem) and much other confusion about the
species concept and the process of speciation (see
[39]). One must use care in reading the literature
on species because the terms genetic isolation and
reproductive isolation are frequently used inter-
changeably. If reproductive isolation is used, it is
usually in the meaning of genetic isolation.
For these reasons, I proposed a minor, but
significant, modification of the bilogical species
concept of Bock [13], namely that: “A species is a
group of actually or potentially interbreeding
populations which is genetically isolated in nature
from other such groups.” This definition is actually
what was meant by the original proposers of the
biological species concept as can be determined
from their entire analysis. Genetic isolation be-
tween species is achieved by the possession of
intrinsic isolating mechanisms (I I M; see Mayr [4]
pp. 91-109; and see below). Mayr [4] introduced
the idea of the non-dimensional species concept
(=the biological species concept) which is most
fundamental, but has been little appreciated by
evolutionary biologists and systematists. Unfortu-
nately Mayr did not connect the non-dimensional
species concept tightly with evolutionary theory
and did not contrast it sufficiently with the multi-
dimensional species notion which is applied in
systematic practice to species taxa. It cannot be
stressed too strongly that further development of
evolutionary theory can be achieved only with the
non-dimensional species concept, not with the
multi-dimensional species notion or all recognized
species taxa.
The species concept applies only to sexually
responducing organisms. It has no meaning for
asexually reproducting organisms which are not
organized into interbreeding uints. Species taxa
appeared in the history of life at some (reasonably
short) time after the evolution of sexual reproduc-
tion, or possibly in conjuction with the evolution of
sexual reproduction. The necessity for species
results from the mechanisms of meiosis (the shuf-
fling and recombination of existing genetic varia-
tion during gamete formation) and of fusion of two
haploid gametes into diploid zygotes of the next
generation, all of which shuffles and recombines
the genetic material existing in the genotypes of
parental organisms into new offspring genotypes.
If too much variation exists among the genotypes
of the parental organisms, then the possibility of
producing too many non-viable offspring is great.
Species, and hence species boundaries, provide
limits to the amount of genetic variation existing
within each species. The arrangement of sexually
reproducing individuals into discrete species taxa
permits a more efficient subdivision of the environ-
ment by sympatric species [9].
A species, therefore, is a group of individual
organisms which are able to interbreed and which
are genetically isolated from other species. As a
species reproduces itself generation after genera-
tion it forms a phyletic lineage which is the time
line (the history) of the species. A cross-section
through a phyletic lineage at any point in time
represents a species. Cross-sections through the
same phyletic lineage at different times represent
species. But it is a non-question to ask whether
these different time slices of a phyletic lineage (=
species) represent the same species or different
species. In theoretical considerations, limits can-
not be placed on a species along a phyletic lineage
because any such limits would necessitate drawing
a boundary between one species and its successor;
such boundaries would imply an evolutionary
change “between species” other than the observed
gradual change “within species” which has never
been demonstrated. Therefore, if species can not
be delimited along a phyletic lineage, it is not
possible to speak of the origin or the brith of a
species, nor is it possible to speak of the age of a
species. All existing species are of equal age, or in
other terms, all species are ageless. And it is not
possible to speak of evolutionary change as being
within the limits of a species or transgressing the
limits of a species; evolution along a phyletic
lineage never results in a new species and hence
never passes a species limit regardless of the
amount of phyletic evolutionary change that has
taken place [12, 13].
700 W. J. Bock
KM 7
Species and speciation are not irreversible as
assumed by some workers. Good species taxa may
evolve and exist sympatrically for a period of time,
but then may start to interbreed, exchange genetic
mateiral and finally reunite into a single species
[13]. An excellent example is the towhees (Pipilo
erythrophthalmus and Pipilo ocaci) found in the
central Mexican highlands [15] which started to
hybridze some hundreds of years ago and have
become a single genetic-reproductive-ecological
unit over most of their range. Such subsequent
interbreeding and merging of formally good spe-
cies depends on the nature of the I I M previously
separated them.
C) Phylogenetic species concepts
Herein I group several diverse species concepts
proposed under the headings of the evolutionary,
phylogenetic and cladistic species concepts [16-
18]. Regardless of other aspects of the definition
for these species concepts, each includes some
reference to a reasonable time dimension for the
7 ———lime T
Time T,
Fic. 1. Schematic diagram illustrating the
concept of the phyletic lineage, which is a
species taxon reproducing itself genera-
tion after generation through time.
Change in the species with respect to time
is phyletic evolution. A cross-section
through the phyletic lineage at any point
in time is a species. Cross-sections at
E different points in time are neither the
fs same species of different species as no
species boundary can separate them; they
are simply different cross-sections of the
same phyletic lineage. (From Bock,
[12]).
species. Hence these definitions confuse the spe-
cies concept with that of the phyletic lineage and as
such are not acceptable. Unless the concepts of
the species and of the phyletic lineage are kept
sharply separated, futher evolutionary theory de-
velopment is not possible. For example, species
can evolve, but phyletic lineages, which represent
the past history of a species, cannot.
D) Morphological species concepts
Morphological species do not now or ever have
existed as a proper theoretical species concept
except as a misnomer for the older typological
species definition. Some workers (e.g., Mishler
and Brandon [10]) have proposed a morphological
species definition in the attempt to advocate a
single species concept for all organisms, including
asexually reproducing forms. In essence, these
definitions do little more than state that the species
is what a taxonomist calls a species. Such recent
efforts to propose morphological species concepts
regardless of the wording of the definition, repre-
The Species Concept 701
sent a confusion between the theoretical definition
of the species concept and guidelines for recogniz-
ing species taxa (multi-dimensional species no-
tions). Unless authors proposing a morphological
species concept also present a sweeping reanalysis
of evolutionary causes and processes, including full
considerations of causes and processes involved in
sexual and asexual reproduction and their bearing
on evolution which support their ideas on the
species, morphological species concepts should be
rejected out of hand.
Recently some cladists (e.g., [17]) have argued
that the species is the smallest recognizable tax-
onomic unit in nature in addition to the usual
cladistic concept that a species is the segment of a
phyletic lineage between two successive branching
points. This idea that the species in the smallest
recognizable taxonomic unit in nature confuses the
species concept with that of geographic variants
within broadly ranging species. It confuses the
theoretical species concept with the practical work
of systematists, forgetting that the species concept
is part of basic theoretical biology, not systematic
practice. And it returns to a concept that is very
similar to it not identical to the typological species
concept which has been discarded along with the
rejection of typology in biological thinking.
E) Recognition species concept
Paterson ([{19] and elsewhere) advocated a spe-
cies concept based on the behavioral and other
means by which members of one species recognize
members of their species and distinguish them
from members of other species. Paterson’s argu-
ment on the invalidity of the biological species
concept confuses completely genetic isolation and
reproductive isolation. In addition, his argument
confuse the basic criteria for a theoretical species
concept with properties of species by which separa-
tion between species is achieved. Several excellent
critiques rejecting the recognition species concept
have been published (e.g., [20, 21]) and need not
be repeated here.
None of these several species concepts, advo-
cated as alternatives to the biological species con-
cept, have a firm foundation in evolutionary theory
and none provide a better understanding of evolu-
tionary units designated by the term species [7-9,
21, 22]. Most fail because they confuse the theore-
tical species definition which falls within the pro-
vince of evolutionary theory and the practical
application of this concept to species taxa in nature
which falls within the province of systematics.
F) Sphere of application
The current species concept (based on evolu-
tionary theory) applies only to sexually reproduc-
ing organisms which is a major difference from the
former typological species concept which applied
to all organisms. This restriction developed with
continued analysis of ideas associated with species
concepts and the decision to focus on genetic
isolation as the major criterion of the species
concept. This was not the only possible develop-
ment of the species concept under evolutionary
theory, but is the one which occurred and hence
the current restriction of the species concept to
sexually reproducing organisms.
Species are not an autonomic consequence of
evolution. Actually there is nothing in basic evolu-
tionary theory which necessitates the existence of
species. Rather they exist in sexually reproducing
organisms as a result of the processes of meiosis in
the formation of gametes and the fusion of ga-
metes in the formation of zygotes of the next
generation, as discuseed above.
G) Major properties of species
Species possess three major sets of properties,
or can be said to represent three different types of
units which have been little discussed prior to Bock
[13]; Mayr [3, 4] mentioned these three properties,
but did not develop them. What is important is
that the evolution of attributes possessed by spe-
cies which are responsible for each of these prop-
erties may occur at different times during the
speciation process and under the action of differ-
ent evolutionary causes. Fully evolved species
possess all three sets of properties completely
developed, but some species which are still in the
process of speciation may not have all of these
properties totally developed. Because most evolu-
tionists have focused so strongly on genetic isola-
tion in their analysis of the species concept and on
the evolution of features associated with genetic
isolation, there has been a serious lack of under-
702 W. J. Bock
standing of the properties of reproductive and
ecological isolation and of their evolution during
speciation. Understanding of these properties
clarifies considerably our comprehension of the
entire process of speciation and of species taxa.
(1) Genetic unit: Members of a species form
a genetic community which is completely geneti-
cally isolated (100%) from other species; that is,
no vertical gene flow occurs between species.
Hence the species is the largest unit in which
genetic recombination can take place. This prop-
erty exists for all species, with the exception that in
a few cases a small amount of vertical gene flow
may occur for a limited period of time between
good species. Genetic isolation is maintained by
the possession of intrinsic isolating mechanisms
(II M) which exist in a variety of forms from
premating to postmating mechanisms (see Mayr
[4] pp. 91-109 for an excellent discussion and
classification of IIM). Any of these diverse
isolating mechanisms completely isolate species
genetically, but they vary in other properties
namely in their ability to isolate these species
reproductively. Since the turn of the century,
evolutionists have concentrated, and quite rightly
sO, on genetic isolation and on IIM in their
analysis of the species concept and have used
genetic isolation as the fundamental criterion for
the species definition. This decision was quite
correct because without genetic isolation, the
other properties of species could not evolve and be
maintained. But evolutionists believed that with
the clarification of genetic isolation between spe-
cies, they have understood all that was essential
about the species concept. Unfortunately this is
simply not the true. Moreover, most workers have
equated I I M and their evolution only with genetic
isolation (generally termed reproductive isolation)
and have not made the distinction between the
evolution of IIM for genetic isolation and the
evolution of II M for reproductive isolation. One
must always keep in mind that a diversity of 11 M
exist and that only some serve for reproductive
isolation. Indeed, even is recent papers [21], the
evolution of genetic isolation is discussed as the
evolution of II M and almost never as the evolu-
tion of genetic isolation which confuses completely
the evolution of some IIM under the action of
mutual selective demands (see below) following
the sympatric phase of speciation.
This confusion leads directly to further mis-
understandings about the “historic adaptationistic
baggage” of the biological speies concept and the
process of speciation [21]. Adaptive aspects during
the process of speciation well covered by Moore
[23] must be reanalyzed with a clear understanding
of the differences between genetic isolation and
reproductive isolation.
(2) Reproductive unit: Members of a species
form a reproductive community which is reproduc-
tively isolated (100%) from other species; that is,
there is no hybridization between species. Species
can be genetically isolated without being reproduc-
tively isolated. Individual members of a species
which attempt to or actually do interbreed with
members of other species incur a reproductive cost
relative to conspecific individuals which do not
attempt to interbreed and hence these interbreed-
ing individuals would be selected against because
they have a lower fitness relative to non-inter-
breeding individuals. Unfortunately most evolu-
tionists have not distinguished between genetic
and reproductive isolation [14], partly because
both types of isolation result from II M. But full
reproductive isolation results only from some
I] M—namely those premating mechanisms pos-
sessing low reproductive costs [categories 1(a) and
1(b) of Mayr’s [4] classification]. And these II M
with low reproductive cost can evolve under selec-
tive demands after species have achieved full gene-
tic isolation.
(3) Ecological unit: Members of a species
form an ecological unit as individual members of
one species have similar ecological requirements
which differ from those of members of other
species. Species can be genetically isolated with-
out being ecologically isolated. Members of spe-
cies which are not ecologically isolated will com-
pete ecologically when sympatric, and those indi-
viduals of the two species which compete ecologi-
cally will have lower fitness relative to conspecific
individuals which do not compete ecologically with
members of other species. Ecological isolation
between species results from the possession of
phenotypic features associated with the ecological
requirements of each species [24-26].
The Species Concept 703
H) Ontological nature of species taxa
Although species taxa will be discussed below,
one aspect, namely the ontological nature of spe-
cies taxa, will be dealt with here. One point is
whether species taxa are “real” or whether they
are just abstractions with the only real units being
the individual organisms. This paper is not the
place to enter a lengthy discussion of the meaning
of reality and the criteria by which various units in
nature are recognized as real. It has been argued
that species taxa are real because the same species
taxa are recognized by groups of people with
totally different traditions and training. Mayr [4]
stated that the natives living in the Arfak Moun-
tains of New Guinea recognize 136 of the 137
species taxa of birds found in that region as
delimited by European systematists who have nev-
er seen these birds alive. Others workers counter
that those native people who may depend less on
birds do not recognize the same species taxa as do
tranined systematists—that the distinction be-
tween species taxa depends on the importance of
these organisms to the life of different human
groups. Nevertheless, it has been demonstrated
time and time again that in local regions, sexually
reproducting organisms are organized, with few
exceptions, into clearly demarcated species taxa
(e.g., Mayr [27]) which are recognized by any
person with the proper training. This agreement in
the recognition of species taxa in local regions
supports sufficiently the reality of species taxa [7].
The second point is whether these species taxa
should be regarded as groups (sets, collections) or
as individuals. This question was raised in the
1970’s by Ghiselin [28, 29]. Hull [30-32], and
Mayr [8] among others who argued that species
taxa do not represent groups, but individuals, with
individual organisms being parts, not members, of
the species taxon. These conclusions are not
completely clear because it is not always definite
that some workers advocating species as indi-
viduals refer to species taxa or to phyletic lineages;
in some cases, the contents of the papers suggest
that the authors allude to phyletic lineages or to a
combination of species taxa and phyletic lineages.
It is argued that the characteristic integration of
individuals is achieved in each species taxon by an
integrated gene pool resulting from gene flow
between all geographic parts of the species taxon
[33]. Serious questions exist whether the gene pool
of a species taxon is sufficiently tightly integrated
as reguired for individuals [13]. The possession of
some integration of the gene pool of a species
taxon is not sufficient for individual status as
certain groups may possess a degree of integration.
What is importance is the tightness of this integra-
tion [13]. Other authors, such as Hull, considered
species taxa as “historical individuals” —that is,
individuals in the sense of historical-narrative ex-
planations (see [34]) which is quite a different
concept from that of individuals in the sense of
nomological-deductive explanations. Other work-
ers (e.g., [35]) countered that species taxa are
proper groups comprised of individual organisms
as members, not parts, but that these groups are
not characterized by typological essences. Analy-
sis of the broad meaning of individuals versus
groups in the philosophy of science suggests
strongly that species taxa are groups, not indi-
viduals, but are groups characterized by nontypo-
logical attributes. Nontypological attributes are
those which can vary in space and time, but are
sufficiently similar to permit recongition of indi-
viduals as members of the group—these are attri-
butes which have been called “family-resemblance
features” existing under the general approach of
“population thinking.”
THE PROCESS OF SPECIATION
A) Evolutionary processes and causes
Before it is possible to discuss the evolution of
species diversity (speciation), it is necessary to
outline the different processes of evolution and to
correlate these with the species and the phyletic
lineages. The process of evolutionary change, in
the broadest sense, can be defined as change in
living organisms with respect to time with the
minmum time period being one generation; hence
evolutionary change is difference observed be-
tween organisms in one generation and those in
descendent generations. Frequently cited defini-
tions of evolution as “genetic changes over time”
are far too restrictive. Evolutionary change con-
704 W. J. Bock
sists of two basic, but quite different processes,
namely phyletic evolution and speciation. Phyletic
evolution can be defined as evolutionary change in
a phyletic lineage. Phyletic evolution results from
(a) the set of causes resulting in genetically-based
phenotypical variation generation after genera-
tion, and (b) selective demands arising from the
external environment. Differences may exist in
the causes and processes of phyletic evolution in
sexually and asexually reproducing organisms, but
these are of no immediate concern to this paper.
Speciation occurs only in sexually reproducing
organisms, and is the multiplication of a species or
the splitting of a phyletic lineage into two or more
lineages. No special causes of speciation exist
other than those acting in phyletic evolution. The
only additional factor occurring in speciation is a
boundary condition in the form of an external
reproductive barrier separating two portions of the
original species into two separate populations and
keeping them genetically isolated during the
Origin of
External
Isolating
, Barrier /
Ancestral Species
c Species B
period in which members of the two populations
could interbreed and exchange genetic material if
they were in contact. During this period, intrinsic
isolating mechanisms may evolve in the two
populations by means of phyletic evolution. It is
clear that phyletic evolution can occur without
speciation, but that speciation can occur only with
accompanying phyletic evolution in one or both
populations isolated by the external barrier.
In sexually reproducing organisms, the unit of
evolutionary change is the interbreeding popula-
tion which may be equivalent to the species if the
geographic range of the species is sufficiently
small. Different populations within species with a
larger geographic range can evolve partly to com-
pletely independently from one another; the
changes in diverse populations are usually con-
nected by gene flow between them, unless, of
course, the different populations are separated by
an external isolating barrier and are in the process
of speciation (see below). The generalization that
Fic. 2. Schematic diagram illustrating the
relationship between speciation (split-
ting of a phyletic lineage into two) and
phyletic evolution. The process of
speciation required the presence of an
external isolating barrier and phyletic
evolution in at least one of the two
lineages. Species A and B are distinct
and separate from one another, but
each is not different from the ancestral
species common to both phyletic
lineages. (From Bock, [12]).
The Species Concept 705
the species is the unit of evolutionary change is
true only in a very rough sense. The phyletic
lineage is never the unit of evolutionary processes
because it is an abstraction composed of the time
line of a species. The phyletic lineage is an
historical representation and hence can no longer
be changed.
It is important to stress most strongly that
although the interbreeding population or the spe-
cies is the unit of evolutionary change, the process,
it is not the unit on which evolutionary causes act.
Causes of phyletic evolution act on individual
organisms, and as a result of these causes including
the reproduction of offspring organisms, evolu-
tionary change occurs.
In addition, it must be stressed that all observed
or postulated phyletic evolutionary changes are
gradual in that they are reducible to the causes
and/or changes observable in populations genera-
tion after generation. These evolutionary changes
may have different rates, but they are all gradual in
the sense just mentioned.
Diverse workers have used other terms, such as
vertical versus horizontal evolution or anagensis
versus cladogensis, rather than phyletic evolution
and speciation. I would recommend against use of
these other terms because they are less clear and
because they involve certain unnecessary connota-
tions which are absent in the terms phyletic evolu-
tion and speciation. Other terms such as phyletic
speciation or vertical speciation have been used
frequently, but in an ambiguous sense for phyletic
evolution; they should be abandoned as they imply
serious misconceptions and erroneous connota-
tions with the process of speciation.
B) Speciation
Speciation, or splitting of a phyletic lineage, is
the process by which two or more descendent
species evolve from a single ancestral species.
Although in most cases, speciation is a dichoto-
mous splitting of phyletic lineages, speciation can-
not be generalized to be always dichotomous as
done by some workers, e.g., cladists. It is entirely
possible to have a simultaneous subdivision of an
ancestral species into three or more geographically
defined portions (different lineages) by external
barriers. The full process of speciation includes
the evolution of the three fundamental properties
—genetic isolation, reproductive isolation and
ecological isolation—in the two or more descen-
dent species, not just the evolution of genetic
isolation as covered in many analyses of specia-
tion. The evolution of each of the these properties
will be discussed separately because the each can
occur independently of the others and as a result of
different sets of evolutionary causes. Moreover
the timing of the evolution of these different
properties is important. Speciation is a popula-
tional phenomenon and is the result of the evolu-
tionary causes underlying the process of phyletic
evolution; hence speciation must be accompanied
by phyletic evolution in at least one of the popula-
tions. There are no distinct evolutionary causes of
speciation, but the additional boundary condition
of an external isolating barrier which divides the
ancestral species into different units and remains in
place sufficiently long to permit independent
phyletic evolution of each of these units. The only
external isolating barrier demonstrated to be suf-
ficient for speciation is a geographical-ecological
one [2, 4] giving rise to distinct allopatric (geo-
graphically separated) populations and leading to
geographic or allopatric speciation. Excluding
speciation by hybridization and polyploidy in some
groups of plants, no evidence exist for suitable
external isolating barriers giving rise to distinct
sympatric populations and leading to sympatric
speciation [4, 26, 36]. Nor is there any evidence
for parapatric speciation in which the two portions
of the ancestral species inhabit different geo-
graphic regions, but have always maintained a
contact with one another as proposed by White
[37]. It is erroneous to ask whether speciation
occurs via geographic isolation or chromosomal
rearrangements, as argued by some workers sup-
porting the concept of parapatric speciation. Both
aspects are involved in those cases where the
intrinsic isolating mechanism results from chromo-
somal differences (see [6]).
The question exists of whether distinct types of
speciation exists within allopatric or geographic
speciation (Mayr, [6]). Diverse workers have
discussed whether speciation occurs by “splitting”
or by “budding”, or whether speciation occus
when the ancestral species is divided into two large
706 W. J. Bock
portions or only when there is a small geographic
isolate—a founder population [4, 33]. Mayr,
among others have argued for a “genetic revolu-
tion” in the founder population (=the founder
principle; Mayr [2] p. 237). The hypothesis of a
genetic revolution postulates that the modification
of the integrated gene pool of the ancestral species
to the new integrated gene pool of the descendent
species occurs at the origin of the founder popula-
tion and is a rapid change. Genetic revolutions
occur by genetic drift in small populations in which
only a small portion of the genetic variation of the
ancestral species is present in the founder popula-
tion [5, 33, 38, 39]. No question exists that phyletic
evolutionary change can be much faster in small
populations, but serious doubt still exists on the
role and importance of the founder principle in the
process of speciation. No one has demonstrated
the high degree of integration of the gene pool of a
species as postulated by Mayr or the ways in which
this “integrated gene pool” can modify over time.
It has not been shown that the only way the
“integrated gene pool” of a species can change is
by genetic revolutions and no one has demon-
strated the workings of a genetic revolution ex-
perimentally in spite of numerous attempts. Nor
has anyone demonstrated the existence of the
“closed genetic system” of species which is mod-
ified only during speciation [38].
Because speciation is more than the evolution of
genetic isolation as will be discussed below, but
also involves the evolution of reproductive isola-
tion and ecological isolation both of which take
place largely after the establishment of sympatry
between the descendent species, additional doubt
exists on the role of the founder principle and
associated concepts such as “punctuated equilib-
rium” as advocated by Eldredge and Gould [40].
C) The speciation process
A full episode of the speciation process begins
with the appearance of an external isolating bar-
rier—a geographic-ecological one in the case of
geographic speciation—and ends with the full
achievement of reproductive and ecological isola-
tion in addition to genetic isolation. This episode
can be divided into two portions, namely (1) the
initial allopatric phase and (2) the subsequent
sympatric (or neosympatric) phase [12, 13]. It
must be stressed that the speciation process is not
completed with the disappearance of the external
isolating barrier and the re-establishment of sym-
patry of the two newly genetically isolated species
as is ususally assumed in discussions of speciation.
Rather this is just the end of the first phase and
important changes take place during the subse-
quent sympatric phase. The same sequence exists
in speciation by hibridization and polyploidy in
which the evolution of genetic isolation is achieved
in a single sexual generation and hence the need
for the alloptric phase is eliminated.
(1) Evolution of genetic isolation: With the
establishment of the external isolating barrier, the
separated populations can undergo independent
phyletic evolution. Intrinsic isolating mechanisms
serving for genetic isolation may appear fortuitous-
ly as pleiotropic consequences of these phyletic
evolutionary changes during the initial allopatric
phase of speciation. Which isolating mechanism
appears and when it appears are strictly chance-
based. Moreover, the evolution of these II M are
never the result of their being favored as genetic
isolating mechansims by selective demands arising
from the external environment. For speciation to
continue, genetic isolation must be 100% complete
by the end of the initial allopatric phase when the
external isolating barrier disappears and the two
populations can expand their geographic ranges
and become sympatric. If genetic isolation is less
than 100% effective, then the members of the two
populations will hybridize and exchange genetic
material [23]; Moore’s excellent analysis must be
read carefully as he is considering genetic isolation
only, not reproductive isolation. The only out-
come of sympatry of two populations with less than
100% genetic isolation is increased hybridization
and subsequent merging of two populations. The
speciation process will come to a halt and the two
populations will continue to exist as a single spe-
cies with the steepness of the secondary zone of
integration between them depending on many
factors. No theoretical argument or empirical
evidence supports the oft-claimed idea [41] that
selective demands from the external environment
will disfavor hybrid individuals and lead to greater
and greater genetic isolation until the level of
The Species Concept 707
Sympatric
Phase
Time
Allopatric
Phase
Onset of
Speciation ~~
Termination of
Speciation
a)Reduction of Competition
b)Reduction of cost of
Geographical
Barrier
reproduction
Initial Overlap
Origin of
Intrinsic Isolating
Mechanism
Subspeciation
Geographical
Spread
Evolutionary Divergence
Fic. 3.
Schematic diagram to show the divergence of the two phyletic lineages during a speciation episode. Retes of
evolutionary change and resulting divergence are low during the period of subspeciation and somewhat higher
during the allopatric phase after the appearance of the geographic-ecological barrier. The rate of divergence
increases sharply after the external barrier disappears and the two species have become sympatric.
Mutual
selective demands pushing the divergence during the sympatric phrase arise from exclusionary species interactions
between members of the two species.
terminates. (From Bock, [12]).
100% genetic isolation is reached [23].
The two populations having achieved sympatry
and possessing 100% genetic isolation with respect
to one another have reached the level of the
species level based on the biological species defini-
tion. Such populations will be recognized as
different species taxa. But the process of specia-
tion is not yet complete.
(2) Evolution of reproductive isolation: As
Speciation comes to an end when this period of rapid divergence
noted earlier reporductive isolation differs from
genetic isolation although these properties of spe-
cies have been confused by most evolutionists.
Both reproductive and genetic isolation are
achieved by the possession of I I M, but not always
the same ones. All II M achieve genetic isolation,
but not all [1M achieve reproductive isolation.
IIM (see Mayr [4] p. 92) differ considerably in
their reproductive cost (see Bock [12] p. 35; [13] p.
708 W. J. Bock
43). If one examines the excellent classification of
IIM presented by Mayr, the listed mechanisms
vary from those possessing very low reproductive
cost [mechanisms 1(1) and 1(b)] to those posses-
sing very high reproductive cost [mechanism 2(d)].
Basically only seasonal, habitat and ethological
isolation [mechanisms 1(a) and 1(b)] achieve com-
plete reproductive isolation between species.
The evolution of reproductive isolation may
begin during the initial allopatric phase with the
appearance of the original IIM and sometimes
may even reach the 100% level by the time of the
disappearance of the external isolating barrier.
But this situation is rare. The more usual case is
that given 100% genetic isolation between the two,
newly sympatric populations, mutual selective de-
mands by members of each species on the other
will favor the evolution of additional 11 M with
lower and lower reproductive costs until the two
species become fully reproductively isolated.
Genetic isolation does not increase, but remains at
100% as selective demands favors the evolution of
I 1M of lower and lower reproductive costs. Re-
productive isolation increases as do morphological
differences between the two newly evolved spe-
cies. This has been discussed as strengthening the
IIM or strengthening the secondary isolating
mechanisms ([4] pp. 552-3) which is valid, but is
not sufficiently clear without making a sharp dis-
tinction between genetic isolation and reproduc-
tion isolation. This point is completely confused
by many evolutionists (e.g., Coyne, et al. [21] p.
195) who state that they know of no earlier work-
ers who claimed that ethological isolating mecha-
nisns are selected for and perfected during specia-
tion. This is in error because I have done so [12,
13], pointing out that such isolating mechanisms
are selected for and perfected during the sympatric
phase of speciation to increase reproductive isola-
tion, not to increase genetic isolation.
(3) Evolution of ecological isolation: At the
time that newly evolved species re-estrablish sym-
patry with the disappearance of the external isolat-
ing barrier, they are generally rather similar ecolo-
gically. Some ecological differences may have
evolved during the allopatric phase of speciation,
but if so, they are generally small [24, 25]. Lack
discussed the several different possibilities of eco-
logical interaction between newly evolved after the
onset of sympatry and the evolutionary consequ-
ences. Members of these species will generally
compete ecologically with one another and exert
mutual selective demands on one another once
they overlap geographically and ecologically. The
result in many cases will be increased divergence
between the newly evolved species in those fea-
tures associated with ecological requirements until
the level of competition drops very low.
Morphological divergence between descendant
species results largely from the evolution of repro-
ductive and ecological isolation as the result of
mutual selective demands exerted by members of
both species on one another following the estab-
lishement of the secondary sympatric phase of
speciation. The final end of the speciation episode
occurs when morphological divergence between
the two species ends or slows down to a very small
rate with the disappearance of the mutual selective
demands exerted by members of the two newly
evolved species on one another. The fact that
much to most of the differences between descen-
dant species evolves during the secondary sympat-
ric phase places doubt on such concepts as the
genetic revolutions and punctuated equilibrium.
SPECIES—THE TAXONOMIC UNIT
A) Recognizing and cataloguing diversity in na-
ture
A primary task of the systematicst is to describe
the observed diversity of living organisms in a
system which will permit an efficient understand-
ing of their atrributes. Many possible systems exist
for arranging these organisms into primary units.
The basis for recognizing species taxa has varied
considerably during the history of biological re-
search, but has generally reflected relatively close-
ly the current theoretical species definition and
changed with modifications in this definition. Cur-
rently, the species definition chosen reflects most
closely the evolutionary history of organisms.
Nevertheless, application of the theoretical species
definition to units (=species taxa) in nature has
never been simple, and a considerable degree of
judgment always had to be used. This was true
The Species Concept 709
under the typological species definition as earlier
systematists agonized over the question of how
distinct a variety of a species had to be before it
could be considered a distinct species. Under the
biological nondimensional species definition, prob-
lems developed immediately as species taxa were
traced further and further in space and time from a
single locality. Many arbitary decisions have to be
made as no absolute standards exist for the recog-
nition of species taxa. Hence, Mayr ([4] p. 19)
coined the multidimensional species notion to deal
with the delimitation of species taxa in space and
time and to distinguish species taxa from the
theoretical concept of the species.
B) Species taxa
Species taxa in sexually reproducting organisms
recognized by taxonomists should correspond as
closely as possible to the non-dimensional species
definition, and hence consist of populations which
actually or potentially interbreed (exchange
genetic material) in nature. The actual or potential
ability of interbreeding is judged on the dual bases
of morphological similarities of organisms within a
species taxon and of gaps between possible species
taxa. Hence the degree of morphological similar-
ity in a Series of actually interbreeding populations
is used as the criterion for deciding on the species
status of isolated populations in which actual inter-
breeding cannot be observed directly. Such
criteria must be used with great care and judge-
ment as rigid standards cannot be established
without running into the danger of a typological
approach. Thus in various biochemical compari-
sons of populations developed during the past two
decades, one must avoid the temptation of estab-
lishing fixed degrees of biochemical differences for
subspecific status, specific status, etc., which can
easily lead to typological thinking.
The second problem is reaching decisions on the
meaning and recognition of species taxa in asexual-
ly reproducing organisms. Clearly such organisms
are not arranged into species taxa similar to those
found in sexually reproducing organisms. Yet
systematists are faced with the problem of describ-
ing and arranging the existing diversity observed in
all organisms, be they sexual or asexual. Begin-
ning at least two hundred years before the accept-
ance of evolutionary theory, the primary units
used in describing this diversity have been called
species. And starting with Linnaeus in the mid-
18th century, the nomenclatural system used in
describing this diversity has been based on species
taxa. Novertheless, under evolutionary theory it is
not possible to formulate a single theoretical spe-
cies concept which is applicable to all organisms as
has been advocated by a number of workers (e.g.,
[10]). Whatever species taxa are recognized for
asexually reproducing organisms, these species
taxa are fundamentally different from species taxa
recognized in sexually reproducing organisms
which are based on the biological species concept.
It is only by tradition and convention that these
primary units recognized by systematists in both
sexually and asexually reproducing organisms are
called species and are considered within systema-
tics to be the same.
Because no theoretical concept exists as the
foundation for species taxa in asexually reproduc-
ing organisms, no unambiguous foundation exists
for criteria on which to recognize species taxa in
these organisms. The best possibility is to reach
some arbitrary decision on criteria for the arrange-
ment of the diversity observed in individual asex-
ual organisms into species taxa. Basically what
systematists have done was to obtain a measure of
the morphological differences observed in phe-
notypic features of sympatric species taxa in sex-
ually reproducing organisms which are associated
with ecological isolation in these species. This
degree of morphological difference was then used
as the criterion for distinguishing species taxa in
asexually reproducing organisms. Justification for
this procedure comes from the argument that
sympatric species taxa in sexual organisms serve to
subdivide the local environment in such a way that
the degree of variation in each species taxon is not
too great to preclude the formation of ecologically
viable offspring at each generation. One can
argue, and completely correctly, that this degree of
variation possessed in species taxa of sexual organ-
isms is governed by the processes of recombination
of genetic material during gamete formation and
their fusion into zygotes, and that this degree of
variation has nohting to do with the amount of
variation contained in species taxa of asexual
710 W. J. Bock
organisms. Therefore one could argue, and quite
rightly, that applying a measure of ecological dis-
tinctiveness observed in sexual species taxa to
asexual organisms has no justification. But if this
position is accepted, then no fundation exists for
any standards on which to recognize species taxa in
asexual organisms, and the recognition of such
species taxa would become completely arbitrary.
Therefore anything goes and the usefulness of
species taxa in asexual organisms would disappear.
The application of a standard based on ecological
differences in sympatric sexual species taxa to
asexual organisms results at least in species taxa
which have ecological meaning and therefore con-
siderable evolutionary meaning in terms of selec-
tive demands exerted by members on one species
taxon on others.
C) Problems of geographic distribution
Species taxa, both of sexual and of asexual
organisms, often have a broad geographic distribu-
tion, one that greatly exceeds the normal dispersal
abilities of individuals during their lifetime. En-
vironmental conditions generally vary over this
geographical range resulting in diverse selective
demands acting on local populations of the species
and hence in geographic variation. Almost all
species taxa with a broad geographic range show a
certain (often considerable) degree of geographi-
cal variation. Because the pattern of change in
environmental factors is not gradual and because
there may be gaps in the geographic distribution of
species taxa, a particular species taxon may be
subdivided recognizable, geographically
arranged subunits, or subspecies (geographical
races). Problems exist when these geographic
subunits are not in direct contact and it is not
possible to determine whether members of the
different units can actually interbreed in nature.
Or when the end forms of a long series of geo-
graphic units meet in nature and do not interbreed,
into
as in the members of the Larus argentatus com-
plex. The taxa argentatus—schistisagus—fuscus
form a continuous ring of interbreeding popula-
tions throughout the Holarctic Region with the
two end fuscus—
coexisting in northern Europe without interbreed-
ing (see Mayr [2] pp. 180-185). Members of the
forms—argentatus and
end forms of the Rana pipiens complex in eastern
United States do not produce viable zygotes in the
laboratory [23, 42]. The central question is
whether gene flow can actually or potentially occur
in nature between the geographic populations of a
species; hence the definition of the multi-
dimensional species notion “as groups of popula-
tions that actually or potentially interbreed with
each other.” ([4] p. 19).
Recently many cladists have advocated a species
concept based on the idea that the species is the
smallest taxonomically recognizable unit in nature
(Cracraft, [17]). Under this concept, each of these
geographical races, especially those in which geog-
raphic gaps separate the ranges of neighboring
races, whould be consideres as different species.
This species concept should be rejected as it re-
turns us to the typological species concept of the
last century and earlier before the appreciation of
geographic variation within species taxa and the
development of the subspecies concept. It would
result in species taxa of very different natures, such
as sympatric species taxa and allopatric species
taxa, which would greatly reduce the value of
species taxa in any comparative analysis.
D) Problems of chronological distribution
Even more troublesome than geogrpahic dis-
tribution is the time distribution of species taxa.
The definition of the species concept indicates that
the species has no time dimension and that there is
no way to distinguish between different species
along a phyletic lineage. Yet practical application
of the multidimensional species notion to fossil
material suggests that recognition of species taxa
along a phyletic lineage is a necessary part of
describing the diversity of organisms existing in the
past and found in the fossil record. Because all the
information a paleontologist can obtain from fossil
specimens are morphological similarities and dif-
ferences between them, decisions on the recogni-
tion of species taxa must be based on analogy from
the morphological variation in recent taxa. Hence
the species taxa of fossil organisms are estimations
at best, even for fossils of presumably sexually
reproducing organisms. Little is gained from ex-
tensive arguments about the limits of species taxa
of fossil organisms. Generally a paleontologist will
The Species Concept
include fossil specimens over a time range in a
single species taxon if they are morphologically
similar, that is, do not shown any phyletic evolu-
tionary change. Gaps in the fossil record are used
as convenient points of delimitation between differ-
ent species taxa arranged in time. Again, such
recognition of species taxa in the fossil record is
only a practical aspect of systematical study of
fossils and must not be used as the basis for further
development of theory. Application of the evolu-
tionary or phylogenetic species concept to with
similar fossil specimens over a reasonable time
dimension is simply not valid. Moreover, it is
difficult to impossible to use species taxa of fossil
organisms to test theoretical concepts about evolu-
tionary theory, such as support for ideas such as
punctuated equilibrium.
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ZOOLOGICAL SCIENCE 9: 713-718 (1992)
The Emetic Response of Urodele Amphibians
Tomio Narrou! and RIcHARD J. WASSERSUG2
'Department of Biology, Shimane University, 1060 Nishikawatsu, Matsue,
Shimane 690, Japan, and *Department of Anatomy and Neurobiology,
Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada
ABSTRACT— Although it is well established that adult anurans can vomit, the one previous study with
a urodele (Triturus) suggested that salamanders lack an emetic response. Using a salamandrid, Cynops
pyrrhogaster, and a hynobiid, Hynobius nebulosus, we reexamined the possibility of emesis in urodeles.
Apomorphine hydrochloride did not induce ejection of gastric contents in C. pyrrhogaster, while a small
number of H. nebulosus showed gastric evacuation at high dosages of this emetic. Copper sulfate and
antimonyl potassium tartrate, in contrast, caused vigorous vomiting in both species. Emetic behavior
was similar in both species with both drugs. The urodeles ejected gastric contents only after substantive
bending or twisting of their torso. This suggests that emesis in salamanders required elevation of
intra-abdominal pressure by contractions of axial and abdominal wall muscles, as it does in anurans.
However, unlike in frogs, the esophagus and stomach do not prolapse outside the mouth when
© 1992 Zoological Society of Japan
salamanders vomit.
INTRODUCTION
The ability to vomit is one of the more sophisti-
cated protections that any animal can have to
toxins in ingested food. Yet not all animals can
vomit. Rats, for example, do not vomit, whereas
cats do. Studies of vomiting in amphibians have
established that adult anurans have a well de-
veloped emetic response [1-5]. Compared to what
is known about anurans, emesis in urodele amphi-
bians has received little study. Mellinger [5]
claimed that the newt Triturus (Salamandridae)
did not vomit, when exposed to apomorphine, and
that single observation has been cited [6] and
miscited [7] several times over the last century.
Given the broadly similar diets of anurans and
urodeles, it would be surprising if Mellinger’s
observation were true for all salamanders. Mellin-
ger’s negative result for Triturus could be the result
of the procedure that he used to induce and
observe emesis. It would certainly be premature to
generalize his conclusion to all urodeles. Given
this background, the question remains: Do any
Accepted April 13, 1992
Received January 22, 1992
" To whom correspondence should be addressed
urodeles possess the ability to vomit and, if they
do, how similar or different is their emetic be-
havior to that of anurans? Our present study
focuses on these questions.
MATERIALS AND METHODS
The salamanders Cynops pyrrhogaster (Sala-
mandridae), weighing 1.9-6.3g, and Hynobius
nebulosus (Hynobiidae), weighing 3.2—7.4 g, were
used as experimental animals. They were field
collected on the outskirts of Matsue, Japan, and
maintained in the laboratory for up to three weeks
on a diet of chopped earthworm, Eisenia foetida,
fed to them twice weekly. Except for a small
number of H. nebulosus, all animals were used
only once.
In order to determine if salamanders can vomit,
three emetics were assessed: apomorphine
hydrochloride (Sigma, St. Louis), copper sulfate
(Wako Pure Chemical, Osaka) and antimonyl
potassium tartrate (Wako Pure Chemical, Osaka).
Apomorphine, which had been dissolved in 0.65%
NaCl solution, was subcutaneously injected into
the dorsum of the proximal hindlimb in dosages of
0.5, 2.0, 5.0, 20, 50, 100, 200, or 300 ug per g body
weight (wet mass). Intraperitoneal injections of
714 T. NaAITOH AND R. J. WASSERSUG
50, 100 or 300 ug/g dosage were additionally
attempted. Copper sulfate was given sub-
cutaneously in the same way as apomorphine, or
orally with catheter into stomach in a dosage of
0.2, 0.4, 1.0 or 2.0 mg per g body weight. Anti-
monyl potassium tartrate was given orally in a
dosage of 0.4 mg/g. The volume of apomorphine
solution injected subcutaneously was 0.01 ml per g
body weight with dosages equal to or below 50 ug/
g but 0.02, 0.04 and 0.06 ml at 100, 200 and 300
ug/g dosages, respectively. Copper sulfate and
antimonyl potassium tartrate were given in a
volume of 0.01 ml per g body weight.
All salamanders were fed their normal labora-
tory diet of chopped earthworm 30 to 60 min
before testing with any emetic agent. Ejection of a
piece of earthworm from the stomach was our
criterion for vomiting. After the emetic was admin-
istered, behaviors of the animals were directly
observed or video-taped (with a JVC-GF-S1000H
video camera) for up to three hours without inter-
ruption. The container was checked again from
ten to seventeen hours later, for the presence of
vomitus. To correlate abdominal contractions with
emetic behavior the proportions of the salamander
torsos were measured with a mm ruler directly
from their images on the video screen before,
during, and after emesis. The length of torso was
measured as the distance between the front and
hind appendages. The width was the maximum
width.
Experiments were done in February and March
for H. nebulosus and from March to April for C.
pyrrhogaster. Room temperature ranged from
19.0 to 23.0°C.
RESULTS
In H. nebulosus, two of five animals vomited in
101.7 min and >180 min following subcutaneous
injection of the 100 ug/g dose of apomorphine
(Table 1). In C. pyrrhogaster, one of six animals
vomited in 40.5 min at the 300 ug/g dosage,
though it was uncertain if this was caused by
apomorphine proper or by the high dose of saline.
In this regard, one out of five control newts
vomited after injection of 0.06 ml/g of 0.65%
NaCl alone, but only after 180 min. At all other
dosages of apomorphine, including the case of
intraperitoneal injection, ejection of gastric con-
tents did not occur. Nevertheless many specimens
of both species, in particular C. pyrrhogaster,
showed an atypically wide gaping of their mouth
for a few seconds, plus strong lateral bending,
ventral flexion, or complex twisting of the body
following apomorphine injections at dosages
higher than 5 ug/g. Furthermore, no subcutane-
ous administration of 0.65% NaCl alone in a
volume of 0.01 or 0.02 ml/g induced any trace of
these same extreme postures in control specimens.
Administration of copper sulfate, in contrast to
apomorphine, dramatically induced vomiting in
both species (see Table 1). An 0.65% NaCl admi-
nistration was not effective. Cynops pyrrhogaster
reacted slightly quicker than H. nebulosus to orally
administered copper sulfate.
At comparable dosages, C. pyrrhogaster tre-
sponded much quicker to oral than subcutaneous
administered copper sulfate. Hynobius nebulosus,
however, did not show a similar difference in its
response time to copper sulfate that could be
associated with the route of administration.
Although less extensively tested, antimony] potas-
sium tartrate also proved to be an effective emetic
(Table 1). Oral administration of this agent in-
duced vomiting in all animals tested in both spe-
cies.
When emesis occurred, the behaviors associated
with ejection of gastric contents were the same in
both species and did not vary with the drug used or
its pathway for administration. Ejection of gastric
contents always occurred in a surge associated with
one to several episodes of bending or twisting of
the torso. In the extreme, the salamanders coiled
into a circular, sigmoidal or reversed-sigmoidal
posture (Figs. 1, 2 and 3). During the contortions
associated with emesis the torso of C. pyrrhogaster
shortened by an average of 12.8% (N=5, range
7.3-15.8%). In contrast, the maximum width of
the body increased on average by only 3.6% (N=
5, range 0-8%). Thus the animals were becoming
shorter without a proportionate expansion of
cross-sectional area to maintain constant volume.
Prolapse of the esophagus or stomach outside the
mouth was never detected. The actual vomiting
act took from about one to eighty sec depending
Emesis in Urodeles 715
TABLE 1. The responses of salamanders to various emetic drug treatments
Cynops pyrrhogaster Hynobius nebulosus
No. vomited Reaction No. vomited Reaction
No. tested times (min)* No. tested times (min)*
Apomorphine HCI**
0.5 ug/g S.c. 0/6
2 0/6
5 0/7 0/5
20 0/8
50 0/6 0/5
100 0/6 2/5 101.7, >180
200 0/6
300 1/6 40.5
Copper Sulfate
0.2 mg/g s.c. 1/3 >180 3/3 38.9+11.8
1.0 mg/g s.c. 2/3 62.9, >180
2.0 mg/g s.c. 3/3 42.0 (n=2), >180
0.2 mg/g p.o. 3/3 26.5+3.2 4/4 31.4+10.6
0.4 mg/g p.o. 2/2 42.1, 6.6
2.0 mg/g p.o. 3/3 4.4+1.9 3/3 13.1+5.0
Antimony! potassium
tartrate
0.4 mg/g p.o. 4/4 1.6+0.3 (n=3), TG 138.0+19.5 (n=6),
>180 (n=1) >180 (n=1)
* Mean+SE given for all animals that responded in less than 180 minutes.
** Intraperitoneal injections of 50, 100, or 200 ug/g did not induce vomiting in any of the 2-3 C.
pyrrhogaster which received them. Note: s.c.=subcutaneous, p.o.=per oral.
*** Includes 4 animals previously used in apomorphine tests.
3cm
Fic. 1. Bending posture during vomiting in salamanders. Left: C. pyrrhogaster; right: H. nebulosus. Arrow
indicates vomitus just appearing. Left and right correspond to Fig. 2, D and Fig. 3, C, respectively. (Images
taken directly from videofilm using Sony Multiscan Video Printer UP-930).
716 T. NaAITOH AND R. J. WASSERSUG
444
O
3.5
2cm
Fic. 2. Vomiting behavior in C. pyrrhogaster induced
by orally administered copper sulfate at a dose of 2.0
mg/g body mass. A . Resting state, 50.0 sec before
the start of the vomiting act. B through F=1.2, 3.5,
4.41, 4.44, 4.8 sec, respectively, after the start of
vomiting. Vomitus (stippled) outside the mouth
appears first in D to the left of the head. It is
completely ejected by F. Note the characteristic
bending of the body in D through E. Drawn from a
videofilm.
-—4.0 sec
on the specimen and the concentration of the
emetic administered. Often episodes of weak
bending or twisting were repeated before culmi-
nating in vomiting and gastric content ejection.
DISCUSSION
Apomorphine and copper sulfate are well estab-
lished emetics in mammals [9]. The former is
known to act centrally, while the latter, centrally
and peripherally depending on how it is adminis-
tered [10]. Antimony! potassium tartrate is also an
effective emetic in mammals [9, 11].
In our previous study surveying the vomiting
response in ten species of anurans [8], Xenopus
laevis was typical for frogs in its sensitivity to
apomorphine hydrochloride. Twenty ug/g and 50
ug/g were effective in Xenopus, whereas Rana
Japonica and Rana ornativentris were less respon-
sive to apomorphine even at dosages of 50 and 100
ug/g body mass. In contrast only two urodele
studied here ejected its gastric contents after apo-
morphine administration at these dosages, though
gaping of the mouth and bending of the body
commonly occurred in both species. Thus, these
amphibians are an order of magnitude or more less
sensitive to this agent than are carnivorous mam-
mals on a zg per g body mass basis. However, the
important point is that urodeles unequivocally
vomit in response to relatively low doses of copper
sulfate and antimonyl potassium tartrate. Mellin-
ger [5], who seems to be the only author to
previously explore emesis in a urodele, concluded
that newts (in his case Triturus) could not vomit.
Our present results with another salamandrid
genus, as well as a hynobiid, do not support this
conclusion.
The response of urodeles to both copper sulfate
Fic. 3. Vomiting behavior in H. nebulosus induced by
copper sulfate administered orally at a dose of 0.4
mg/g body mass. A. Resting state, 4.0 sec before
the start of the vomiting act. B through H=0.8,
31.3, 32.8, 35.0, 63.1, 69.4 and 72.3 sec, respective-
ly, after the start of vomiting. Vomitus (stippled)
first appears in C and is completely ejected from the
mouth by H. Sigmoidal posture (C) and twisting of
the body (C to E) are shown. In D, abdominal
contraction is marked. Drawn from a videofilm.
Emesis in Urodeles WAG
and antimony! potassium tartrate leads us to tenta-
tively suggest that most, if not all, amphibians
possess an emetic capability. This would be consis-
tent with the fact that salamanders and frogs both
feed on relatively large and potentially toxic prey,
with little or no oral processing. In mammals,
carnivores that swallow large boluses of unmasti-
cated food have a strong emetic response, whereas
rodents, which extensively process and reduce
food within their oral cavity exhibit a taste aver-
sion response (see Fox, 1990 and other references
cited therein [12]), but not the ability to vomit. It
remains to be seen whether caecilians, which feed
on quite different prey and in a quite different
manner from frogs and salamanders [13], can
vomit.
Both subcutaneous injection and oral adminis-
tration of copper sulfate were potently effective in
inducing vomiting. The neurobiology of emesis in
amphibians remains unstudied, but on pharmaco-
logical grounds it seems similar to that of mammals
described by Wang and Borison [10].
One of six newts vomited after subcutaneous
injection of apomorphine at a very high (300 g/g)
dose. However, this may not be the effect of
apomorphine, since vomiting was also observed in
one instance after a simple injection of the same
volume of NaCl solution. It should be noted that
relatively low doses of apomorphine, but not
saline, induced marked behaviors such as twisting
of the body and gaping of the mouth, without
ejection of gastric contents. If such behaviors are
indicative of retching, the neural mechanisms of
apomorphine-induced vomiting may differ slightly
from that of vomiting induced by systemically
administered copper sulfate.
The most conspicuous feature of vomiting in
salamanders is the preparatory bending and twist-
ing of the torso. These axial movements, in
conjunction with tonic contraction of the muscles
of the abdominal wall, elevate intra-abdominal
pressure. Our observations suggest that salaman-
ders raise intra-abdominal pressure by bending
and twisting movements of the torso which com-
press the abdominal cavity. Previous studies have
shown that elevation of intra-abdominal pressure
is necessary to eject stomach contents in frogs [2—
4]. Similar elevation of abdominal pressure seems
necessary for emesis in urodeles as well. In this
regard, emesis in amphibians is similar to that of
mammals and is not a visceral response as simplis-
tic as (reverse) antiperistalsis.
Prolapse of stomach, however, which is common
during emesis in anurans, was not observed in
urodeles. This is likely due to the gross structural
differences, such as the proportionately longer and
narrower esophagus of salamanders and their
longer bodies overall.
This work was conducted following the “Guid-
ing Principles for the Care and Use of Animals in
the Field of Physiological Science” set by the
Physiological Society of Japan.
ACKNOWLEDGEMENTS
We thank Mr. Masahiro Kirihara and Miss Motoko
Matuura for maintaining the animals in the laboratory
and their help in conducting the experiments. This work
is part of a joint program on “Visceral Functions in
Amphibians” supported by grants from the International
Scientic Research Program of the Ministry of Education,
Science and Culture (Japan) and the Natural Science and
Engineering Research Council (Canada).
REFERENCES
1 Tokita, K., Iwasaki, S. and Yui, F. (1953) On the
Opening motion of mouth and vomiting of frogs after
the digitalis preparations and on their mechanisms.
Folia Pharmacol. Japon., 49: 138-142.
2 Hukuhara, T., Naitoh, T. and Kameyama, H.
(1973) A peculiar phenomenon, the prolapsus of the
esophagus-stomach, observed in the vomiting of the
frog. Jpn. J. Smooth Muscle Res., 9: 1-8.
3 Naitoh, T., Nakai-Kameyama, H. and Hukuhara,
T. (1981) The effect of denervation of abdominal
musculature on the prolapse of esophagus and sto-
mach in the vomiting of frogs. Mem. Fac. Sci.
Shimane Univ., 15: 57-63.
4 Naitoh, T., Wassersug, R. J. and Leslie, R. A.
(1989) The physiology, morphology, and ontogeny
of emetic behavior in anuran amphibians. Physiol.
Zool., 62: 819-843.
5 Mellinger, C. (1881) Beitrage zur Kenntniss des
Erbrechens. Arch. Ges. Physiol. Pfltiger’s, 24: 232—
245.
6 Reeder, W. G. (1964) The Digestive System. In
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Academic Press, New York, N.Y. pp. 99-149.
7 Nigam, H. C. (1977) Vomiting response in the
Indian frogs Rana tigrina (Daud) and Rana cyanoph-
10
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lyctis (Boulenger). Curr. Sci., 46: 791-792.
Naitoh, T., Imamura, M. and Wassersug, R. J.
(1991) Interspecific variation in the emetic response
of anurans. Comp. Biochem. Physiol., 100C: 353-
359.
Borison, H. L. and Wang, S. C. (1953) Physiology
and pharmacology of vomiting. Pharmacol. Rev., 5:
193-230.
Wang, S. C. and Borison, H. L. (1952) A new
concept of organization of the central emetic
mechanism: Recent studies on the sites of action of
apomorphine, copper sulfate and cardiac glycosides.
T. NAITOH AND R. J.
11
12
13
WASSERSUG
Gastroenterology, 22: 1-12.
Koppanyi, T. (1930) Studies on defecation, with
special reference to a medullary defecation center.
J. Lab. Clin. Med., 16: 225-238.
Fox, R. A. (1990) Investigating motion sickness
using the conditioned taste aversion paradigm. In
“Motion and Space Sickness”. Ed. by G. H. Cramp-
ton, CRC Press, Boca Raton, pp. 105-121.
Bemis, W. E., Schwenk, K. and Wake, M. H.
(1983) Morphology and function of the feeding
apparatus in Dermophis mexicanus (Amphibia:
Gymnophiona). Zool. J. Linn. Soc., 77: 75-96.
ZOOLOGICAL SCIENCE 9: 719-724 (1992)
Origin of Serotonergic Innervation of Olfactory Bulbs in the
European Hamster, Cricetus cricetus: An Autoradiographic
Study Using [°H]5-HT Retrograde Labelling
JEAN Luc Miro!, SirviA ARANEDA~
and BERNARD CANGUILHEM
Institute de Physiologie, Faculté de Médecine, Université Louis Pasteur,
67085 Strasbourg Cedex, France and URA CNRS 1332, and *Départment
de Cytologie, Institut des Neurosciences, Université P. et M. Curie. 7,
quai St-Bernard. 75252 Paris cedex 05, France
ABSTRACT — Serotonergic afferents to the olfactory bulb of the European hamster, Cricetus cricetus
were investigated by autoradiography following [*H]5-HT retrograde labelling techniques. Injections of
[PH]5-HT into the main olfactory bulb resulted in radiolabelling of some nerve terminals in the granular
and glomerular layers of the olfactory bulb, and in cell bodies of the median raphe and dorsal raphe. In
the dorsal raphe, labelled cells were located in the dorso-ventral and lateral part of the nuclei. In the
median raphe, labelled neurons were observed more caudally in the medial region. In this nucleus, the
number of radiolabelled cells was a third of those in the raphe dorsalis. A few scattered radiolabelled
cells were observed in the periaqueductal gray. The number of radiolabelled cells in both the raphe
nuclei was not changed when animals were treated with a monoamine oxidase (MAO) inhibitor,
Clorgyline. However, the intensity of labelling was increased in the treated animals. Treatment with
5,7-dihydroxytryptamine abolished the [°H]5-HT retrograde labelling in the olfactory bulb, as well as in
the raphe cell bodies. It is concluded that in the Cricetus cricetus the granular and glomerular layers of
© 1992 Zoological Society of Japan
the ofactory bulbs receive a serotonergic innervation from the median and dorsal raphe.
INTRODUCTION
During the cold season hibernators display bouts
of deep hibernation characterized by a low body
temperature and a reduced basal metabolic rate.
Hibernation is interrupted by spontaneous arousal
and a return to an euthermic body temperature for
a few hours or days. The time course of entrance
into hibernation has only been studied in detail in
few species by monitoring the body or hypotha-
lamic temperature and recording electrical activ-
ity. From these studies two main results have been
obtained. Firstly, the decline in body or brain
temperature is not a passive process but a control-
led one [1]. Secondly, entrance into hibernation is
thought to be an extension of normal slow wave
Accepted April 21, 1992
Received November 12, 1991
1 Deceased on May 27th, 1992
sleep [2]. In certain species it seems that entrance
into hibernation is achieved by way of.serotonergic
system (see ref. in [3]). For example in the
European hamster, Cricetus cricetus, serotonin (5-
HT) concentrations in differents brain areas, espe-
cially in the limbic system, were lower in hibernat-
ing animals than in aroused animals in winter [4].
In addition, electrolytic or pharmacological lesion
of the median but not of the dorsal raphe nucleus
[3] as well as total ablation of the olfactory bulbs
(OB) [5] has led to a suppression of hibernation.
The evidence thus strongly suggests an anatomical
connection between the raphe nuclei and the OB
of the European hamster.
Recently, retrograde axonal transport with triti-
ated transmitters has been developed as a specific
autoradiographic method for tracing pathways in
the CNS [6]. Using this technique [*H]5-HT is
specifically taken up at serotonergic nerve termi-
nals and then transported by retrograde axonal
720 J. L. Miro, S. ARANEDA AND B. CANGUILHEM
transport to their corresponding cell bodies [7-9].
The specificity of this methodology was dependent
upon the concentration of injected [(PH]5-HT, and
has been confirmed using combined studies of
(SH]5-HT retrograde labelling and immuno-
cytochemistry for serotonin [10]. Consequently,
[(>H]5-HT retrograde radiolabelling is now re-
garded as an important technique for studying the
projections of serotonergic neurons of the mid-
brain and medullary raphe in the rat [11]. In view
of the involvement of the median raphe, olfactory
bulb and serotonergic system in the process of
entrance into hibernation, we have investigated
the origin of the serotonergic innervation of the
OB in the European hamster using [*H]5-HT
retrograde labelling.
It has been previously reported that the uptake
[>H]amines by central catecholaminergic fibres is
decreased in hibernating animals [12]. This result
and those reported previously for the serotonergic
system [3, 4, 14-16] suggest that the activity of the
serotonergic neurons could be different between
winter and summer hamsters. In order to test this
hypothesis, we also designed experiments using
retrograde axonal transport with [°H]S-HT in
winter arising hamsters.
MATERIALS AND METHODS
Adult male European hamsters (Cricetus cri-
cetus) were caught at the end of the hibernation
period (April-May) in the field surrounding Stras-
bourg (France). They were kept in individual
cages with free access to food and water. For the
summer experiments 19 hamsters were housed for
one month (July) in a 12h light:12h dark cycle
and constant temperature (20+1°C) before surgi-
cal manipulation. The summer animals were di-
vided into three groups. The first one was a
control groups (n+5) which only received [?H]5-
HT injections. The second group (n+5) was
pretreated with clorgyline, a MAO inhibitor (10
mg/kg), 30 min before the [*H]5-HT injections in
order to prevent the degradation of 5-HT into
5-hydroxyindole-3-acetic acid. The third group (n
+9) was injected with 5,7-DHT (5,7-dihydro-
xytryptamine creatinine sulfate, Regis Chemicals,
8 wg in 2 yl of saline with 0.1% ascorbic acid)
which selectively destroys serotonergic nerve ter-
minals. The 5,7-DHT solution was injected into
the OB at the same stereotaxic cooredinates as the
[>H]5-HT injections six days before.
For the winter experiments, 4 animals were
transferred into a cold room (7+1°C) with total
darkness for one month (January) before the ex-
periment. During this time hibernation was check-
ed daily by visual inspection. Thirty min before
the experiments, the hamsters were pinched in
order to produce an arousal so that when the
[H]S-HT injections began all the animals were
normothermic. Hamsters from these experiments
were not treated with any drugs before the [°H]5-
HT injections.
For the injections of [H]5-HT, animals were
anaesthetised with Nembutal (50 mg/kg) and were
placed in a David Kopf stereotaxic instrument. A
mechanical obstruction of the olfactory diverticle
was then carried out (AP=15, L=1, 2 and DV=
3, 4) in order to avoid difussion of the radioactive
tracer as previously described in the rat by Ara-
neday, ctqysalgauilail: [(H]-serotonin [°H]5-
hydroxytryptamine creatinine sulfate, Amersham,
15 Ci/mol) was injected 20 wCi, 5x 10* M) in one
or both OB (AP=17, L=1 and DV=3) over a
Fic. 1.
injections into the olfactory bulb.
Autoradiograms of the olfactory bulb (A), dorsal raphe (B, C) and median raphe (D) following [*H]S-HT
A) The PHI5-HT injection site in the olfactory bulb was located in the granular layer, where an intense
autoradiographic reaction was observed which decreased in the plexiform and glomerular layers. Scale bar, 80
pm.
B and C) Labelled cell bodies in the dorsal raphe, 24 hours after injections into the olfactory bulb. They were
situated ipsilaterally when the [*H]5-HT was injected into one olfactory bulb. Some radiolabelled cells were
located between both medial longitudinal bundles (mlb). Scale bar, 30 “m.
D) In the median raphe, labelled cell bodies were observed in the mediolateral part of the nuclei and presented a
dense autoradiographic labelling. Scale bar, 45 zm. Note that the silver grains were mainly distributed in the
cytoplasm. The area indicated by arrows is enlarged in the inset. Scale bar, 20 um.
Serotonin and Olfactory Bulbs
in Hamster
722 J. L. Miro, S. ARANEDA AND B. CANGUILHEM
period of 25 min. Twenty four hours later, animals
were re-anaesthetised and the brains fixed by
intra-aortic perfusion using glutaraldehyde (3.5%)
in phosphate buffer (0.2 M, pH7.3). The whole
brain was removed, dehydrated in ethanol and
embedded in parafin-paraplast. Sections (7 ~m
thick) were cut every 50 um. Then coated by
dipping in Ilford KS emulsion (diluted 1:1), ex-
posed for 10-30 days at 6°C, developed in phenisol
(Ilford) and stained with cresyl violet. Light
microscope observations were made using both
bright and darkfield illumination. Radiolabelled
cells were counted in the midbrain raphe and the
Bartlett’s test for homogeneity of variance was
used to determine statistical significance.
RESULTS
Injection site
(H]5-HT injections into the OB of summer
hamsters were characterised by a central zone of
high density of silver grains confined to the granu-
lar layer of the olfactory bulb. The intensity of
this autoradiographic reaction gradually decreased
towards the glomerular layer (Fig.1A), and
caudally towards the anterior olfactory nucleus. A
dense accumulation of silver grains was observed
in the granular, plexiform and glomerular layers
within 1 mm of the injection site. Accumulation of
silver grains was restricted to the neuropile and not
to the cellular components. The mitral tufted and
granular cells were devoid of silver grains.
Following 5,7-DHT treatment, a diffuse auto-
radiographic reaction persisted but the accumula-
tion of silver grains was abolished in all of the
olfactory layers.
Raphe cell bodies
Dorsal and median raphe nuclei of the Euro-
pean hamster are located in the mesencephalon
ventral to the aqueduct. As has been described for
Fic. 2.
Schematic representation of dorsal (DR) and median raphe (MR) and the distribution of labelled cell bodies
(dots) detected by autoradiography following retrograde axonal transport with [SH]5-HT. Brain sections were
taken at 150 ~m intervals to be drawn using a camera lucida. Brain regions were identified with reference to the
rat atlas of Paxinos and Watson (18). 3n: oculomotor nerve, mlb: medial longitudinal bundle, xscp: decussation
of the superior cerebellar peduncle, CG: central gray, Aq: aqueduct, scp: superior cerebellar peduncle, TN:
tegmental nucleus, ml: medial lemniscus, 5 Mo: motor trigeminal nucleus, LTN: lateral tegmental nucleus.
Serotonin and Olfactory Bulbs in Hamster 723
the rat dorsal raphe [12], we also distinguished
four principal regions in the dorsal raphe of the
hamster: the ventromedian, the dorsomedian and
the lateral groups, and the pars caudalis. The
median raphe was situated ventrally to the supe-
rior cerebellar peduncle in the mediolateral posi-
tion. At the rostro-caudal levels, it extended from
the interpenducular nucleus to the caudal levels of
the tegmental nucleus (Fig. 2).
Twenty four hours after the [>H]5-HT injections
into the OB, a number (n=68) of dorsal raphe cell
bodies showed specific autoradiographic labelling
(Fig. 1B-D). Silver grains were distributed in the
cytoplasm and sometimes extended to the de-
ndrites but not to the nuclei. Radiolabelled cells
were observed in the ventral position and scattered
among many non-radiolabelled neurons. At the
rostral level of the superior cerebellar peduncle, a
moderate number of radiolabelled cells were
observed in the medial region of the dorsal raphe.
Caudally, there were more radiolabelled cells dis-
tributed dorsal to and between the medial longitu-
dinal bundle (Fig. 2). A few labelled cells were
also observed in the adjacent central gray.
In the median raphe, the largest number of
retrogradely labelled cells (n=20) were observed
in the ventral position, and situated more caudally
to the labelled cells of the dorsal raphe (Fig. 2). In
both raphe nuclei, radiolabelled cell bodies were
situated mainly ipsilaterally when [*H]5-HT was
injected into one OB (Fig. 1C). The locus coe-
ruleus neurons were not radiolabelled.
Results from the winter experiments indicate
that the number of radiolabelled cells in the dorsal
(n=45+20) and median raphe (n=17+9) were
not statistically different from the summer hams-
ters. The number of radiolabelled neurons in the
dorsal raphe and median raphe of summer ham-
sters was 50+18 and 17+9, respectively.
DISCUSSION
Injection of radiolabelled serotonin into the OB
of European hamsters is followed by a selective
accumulation of radioactivity in some cells of the
dorsal and median raphe. This autoradiographic
labelling depends upon uptake of the radioactive
amine by the serotonergic nerve terminals since
their selective destruction by 5,7-DHT did not
result in a retrograde accumulation in the corres-
ponding cell bodies. Moreover, the lack of label-
ling of the noradrenergic neurons of the locus
coeruleus indicated that this process was selective
to 5-HT neurons. These results on the selectivity
of labelling serotonergic projections are in good
agreement with those obtained in the rat OB-
raphe connections [10]. Using a concentration of
10* M [°H]5-HT, we have also previously demon-
strated specific radiolabelling using a combination
of autoradiography after [*H]5-HT retrograde
transport and immunocytochemistry for serotonin
in both the olfactory and spinal serotonergic pro-
jections [11]. In the present study, inhibition of
the enzyme monoamine oxidase by clorgyline did
not affect the number of labelled neurons indicat-
ing that the [*H]5-HT taken up by the terminals
could be quickly bound or metabolized to another
form in order to be transported retrogradely by
axons as it has been described in the rat [9].
The present findings thus demonstrate a sero-
tonergic innervation of the hamster OB by the
dorsal and median raphe. Radiolabelling was
located in the dorsomedian, ventromedian and
lateral serotonergic cell groups in the dorsal raphe,
and caudally in the median raphe. Moreover,
radiolabelled neurons of both raphe innervate the
ipsilateral OB. These results are in accordance
with previous horseradish peroxidase anterograde-
retrograde tracing [17] and [°H]5-HT retrograde
autoradiographic studies in the rat [8, 11].
Although further studies will be required to deter-
mine the precise anatomical site of serotonergic
innervation of hamster OB structures, it appears
likely from our results that many of the termina-
tions are in the granular and glomerular layers.
In the dorsal raphe, the distribution of sero-
tonergic radiolabelled cells was mainly in the ven-
tral and dorsal position which seems quite similar
to that observed in the dorsal raphe of the rat [12].
In the median raphe, however, labelled neurons
were located more caudally, and were not co-
distributed in the same frontal section of the dorsal
labelled cells as has been previously observed in
the rat using the same methodology [11].
Our previous experiments have suggested that in
the European hamster only specific groups of
724
serotonergic neurons in a small part of the median
raphe are involved in the process of entrance into
hibernation which may or may not be associated
with the OB [3, 5]. Our present results lend some
support to this assumption. If our hypothesis were
true, however, we would expect to find a greater
number of labelled cells in the summer than in the
winter animals indicating increased accumulation
of [PH|5-HT in serotonergic cell bodies before
entrance into hibernation. This was not the case
and further studies should thus be undertaken
using true hibernating hamsters in order to address
this question.
In conclusion, we have demonstrated a sero-
tonergic innervation of the hamster OB by a well
defined population of neurons from the median
and dorsal raphe.
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Heller, H. C. (1977) Sleep and hibernation in
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4 Canguilhem, B., Kempf, E., Mack, G. and Schmitt,
P. (1977) Regional studies of brain serotonin and
norepinephine in the hibernating, awakening or
active European Hamster, Cricetus cricetus, during
winter. Comp. Biochem. Physiol., 57C: 175-179.
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Effects of bulbectomy on hibernation, food intake
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A., Wiklund, L. and Streit, P. (1982) Transmitter
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(1983) Retrograde axonal transport after radioac-
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Magoul, R., Oblin, A., Calas, A. and Araneda, S.
(1988) Serotonergic projections to the spinal cord
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ZOOLOGICAL SCIENCE 9: 725-731 (1992)
Photoperiodic Control of the Determination of Two Different Seasonal
Diphenisms of the Asian Comma Butterfly, Polygonia c-aureum L.
KATSUHIKO ENDO, SATOSHI UENO, MiTSUNORI MATSUFUJI
and YOSHIKI KAKUO
Environmental Biology Laboratory, Biological Institute, Faculty of Science,
Yamaguchi University, Yamaguchi 753, Japan
ABSTRACT—The Asian comma butterfly, Polygonia c-aureum L. exhihits two different seasonal
diphenisms in the morph and adult reproductive activity, both of which are governed by photoperiod
and temperature existing during the post-embryonic development.
In the complete and 8L-16D asymmetrical skeleton photoperiods at 20°C or 25°C, they formed similar
response curves in the incidences of autumn morph and reproductive diapause in adult females.
They required 4 and 5 16L-8D (long-day) days for achieving 50% occurrence of vitellogenic and
summer morph adults, respectively. Critical stages of 16L-8D to 8L-16D (short-day) transfer obtained
on 50% preventions of vitellogenic and summer morph female adults were located on day 0 and on day 2
of the 3rd instar. In contrast, the critical stages of adverse photoperiodic transfer greatly differed
between these two kinds of seasonal diphenisms. The stages were day 3 of the Sth instar in the seasonal
morphs and day 3 of pupae in the reproductive activity.
Physiological mechanisms underlying photoperiodic control in P. c-aureum has been previously
indicated to involve two neuroendrocrine systems secreting summer morph-producing hormone and/or
a factor activating corpora allata in adults. Differential responsiveness of these two neuroendocrine
systems to photoperiods may reflect on the difference in the incidences of the seasonal morphs and
reproductive activity. In addition, the photoperiodic sensitive stage seems to be different with respect to
© 1992 Zoological Society of Japan
these two different seasonal diphenisms.
INTRODUCTION
In a number of insects, diapause and seasonal
polyphenism are governed by environmental fac-
tors, such as photoperiod and temperature they
are subjected to after hatching [1-5].
Pupal diapause and spring morphs of the swal-
lowtail, Papilio xuthus L., are induced by short
days at 20°C. They become photoperiodically
sensitive at around the first larval ecdysis [6]. The
photoperiodic sensitiveness is lost at the middle of
the 5th instar. In the photoperiod-sensitive stage,
they require seven 8L-16D (SD: short-day) days
for the 50% incidences (or require five 16L-8D
(LD: long-day) days for the 50% preventions) of
pupal diapause and spring morph development at
20°C [6].
The Asian comma butterfly, Polygonia c-
aureum L., exhibits two different seasonal diphe-
Accepted April 28, 1992
Received March 11, 1992
nisms, 1.€. seasonal morph and adult reproductive
activity, both of which are determined by photo-
period and temperature they are exposed to after
hatching. Under LD at 20°C, P. c-awreum females
developed into vitellognic/summer morph adults,
whereas those held in SD at 20°C developed into
reproductive diapause/autumn morph adults [7].
Physiological mechanism underlying photo-
periodic control of these two different seasonal
diphenisms involves cerebral factors secreted from
corpus cardiacum and/or corpus allatum in the
early pupal stages [7-9]. Critical stages at which
summer-morph producers (long-day pupae) re-
quire no more brain and/or nervi corporis cardiaci
(I+1I) for summer morph and vitellogenic adult
development lie at about 32 hr after larval-pupal
ecdysis at 20°C in P. c-aureum [10, 11].
Our present study was initiated to determine the
number of long-day required for the occurrence of
summer morph and vitellogenic female adults of P.
c-aureum. Then, the study was extended to deter-
726 K. Enpo, S.
mine the beginning and the end of photoperiod-
sensitive stages in the induction (or prevention) of
these two different seasonal diphenism.
MATERIALS AND METHODS
Animals P. c-aureum collected in the Yama-
guchi City was used. Eggs and larvae were held in
transparent plastic containers of either 695 cm
or 19x13xX5cm and were exposed to complete
photoperiods, whose photophase was changed
from 2hr (2L-22D) to 20hr (20L-4D) at 2-hr
intervals. Rearing containers were placed in
cabinets with constant temperature (20°C or 25°C)
and were illuminated by two 20-W white fluores-
cent tubes which were controlled by a 24-hour
time-switch. In the light period, light intensity was
at about 500 lux to the rearing containers. Larvae
were fed on fresh leves of Humulus japonicus
provided daily.
Classification of seasonal morphs Summer-
morph and autumn-morph females exhibit clear
differences in background color of the wings. The
wing background color of summer morph females
is dark yellow on both sides, whereas that of
autumn morph females is reddish-brown on the
dorsal side and dark brown on the ventral side
[12]. Seasonal morph classification was made only
in females on the basis of the wing background
color.
Criterion for adult reproductive diapause When
fed on 10% sucrose four days after emergence at
25°C, an vitellogenic (reproductive non-diapause)
females accumulate green yolk in oocytes and/or
produce eggs. In contrast, females entering repro-
ductive diapause do not accumulate yolk at the
same period.
Female adults were caught on the day of emerg-
ence, fed on 10% sucrose for 4 days at 25°C and
dissected in 0.9% NaCl to see whether the oocytes
in the ovaries accumulated yolk or not. Female
adults accumulating green-colored yolk in oocytes
were classified as vitellogenic, whereas others were
classified as diapausing.
UENO et al.
RESULTS
Fluctuations of the incidences of autumn morph
and reproductive diapause female adults in the
complete photoperiods at 20°C and 25°C
To assess how the incidences of autumn morph
and reproductive diapause adults fluctuate, groups
of 150-200 larvae were subjected to complete
photoperiods whose photophase was changed de-
pending on larval groups from 2 hr (2L-22D) to 20
hr (20L-4D) at 2-hr intervals at 20°C or 25°C.
Photoperiodic response curves were obtained on
the basis of the percentages of autumn morph and
reproductive diapause adult females at 20°C and
25°C (Fig. la and b).
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Photoperiod
Fic. 1. Photoperiodic response curves of P. c-aureum
two different seasonal diphenisms in the complete
photoperiods at 20°C (a) and 25°C (b). Open and
solid circles show the incidences of autumn morph
and reproductive diapause female adults, respec-
tively.
Seasonal Diphenisms of Polygonia 727
Under shorter than 12 hr photoperiodic regi-
ments at 20°C, all females developed into autumn
morph/reproductive diapause adults with an ex-
ception of 2L-22D group in which 12 summer
morph and 2 (out of 18) autumn morph adults
were vitellogenic. In contrast, all females de-
veloped into vitellogenic/summer morph adults
under longer 14hr photoperiodic regimens at
AIG:
At 25°C, vetellogenic/summer morph adults
appeared in all groups reared under complete
photoperiods examined. Under short-day condi-
tions at 25°C, percentages of autumn morphs
varied in a range from 55% (2L-22D) to 95%
(12L-12D) and 5% to 75% of the autumn morphs
were judged as being vitellogenic. In contrast, all
females developed into vitellogenic and summer
morph adults under longer 14 hr photoperiodic
regimens. Critical photoperiods for the determina-
tion of these two different seasonal diphenisms
were about 13 hr (13.1 hr and 12.7 hr) both at 20°C
and at 25°C.
The results indicate that P. c-aureum used the
same critical daylength for determining both sea-
sonal morphs and reproductive activity, in the
complete photoperiods at 20°C and 25°C. How-
ever, autumn morph development seems not al-
ways to be accompanied by reproductive diapause
at 25°C.
Fluctuation of the incidences of autumn morph and
reproductive diapause adult females in the asym-
metrical skeleton photoperiods of 8L-16D
To clarify how the incidences of autumn morph
and reproductive diapause adults are fluctuated by
a supplementary light pulse at 20°C, groups of
200-250 larvae were subjected to a photoperiodic
regimen of 8L-16D, whose scottophase was inter-
rupted by a supplementary light pulse of 0.5 hr.
The giving time of light pulse was delayed from
light-off of 8L-16D at 1-hr intervals.
All females developed into autumn morph/re-
productive diapause adults under 8L-16D at 20°C.
All or a large part of females were prevented from
exhibiting autumn morph and reproductive di-
apause by a 0.5-hr supplementary light pulse on 3—
6hr after light-off of 8L-16D. However, a light
pulse earlier or later in scotophase was ineffective
where all or majority of females developed into
autumn morph adults, 5% to 30% of which were
vitellogenic (Fig. 2).
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Time of light pulse after dawn (hours)
Fic. 2. Photoperiodic response curves of P. c-aureum
two different seasonal diphenisms in the asymmet-
rical skeleton photoperiods of 8L-16D at 20°C,
where 0.5-hr light pulse interrupted the scotophase
on the time given in the abscissa. Open and solid
circels show the incidences of autumn morph and
reproductive diapause female adults. Open trian-
gles show the incidence of autumn morphs recorded
under the corresponding complete photo-periods at
20°C, redrawn from Fig. 1.
For the 50% prevention of autumn morph de-
velopment, P. c-aureum has two clear critical
photoperiods of 10.5-11.0 hr and 13.0—-13.5 hr in
the asymmetrical skeleton photoperiods of 8L-16D
at 20°C. The shorter critical daylength was almost
the same value as, but the longer one was about
0.5-hr shorter than, the critical daylength of the
50% prevention of reproductive diapause (Fig. 2).
Response curves to the number of 16L-8D (LD:
long-day) days for inducing the development of
summer morph and vitellogenic female adults
To investigate how the effect of LD days is
integrated and represented on the incidences of
summer morph and vitelogenic adult females, 0-
day-old 4th-instar larvae were selected from a
stock culture raised from the egg stage under short
days (SD-8L-16D) at 20°C. The LD-exposure was
performed on 4th-instar or 4th- to 5th-instar larvae
and the length of the exposure was changed from
zero to 7 days depending on the groups.
Any prevention in the incidence of autumn
morph adults was not observed by a LD-exposure
of shorter length than 3 days. But, a few percent of
728 K. Enpbo, S. UENo et al.
the autumn morph adults became vitellogenic.
Percentages of the summer morph adults rose
gradually with the increase of the numbers of
LD-exposure days. Fifty percent of the incidences
of summer morph and vitellogenic adult females
reached by a LD-exposure of five and four days,
respectively. One hundred percents were achieved
by the LD-exposure of seven days (Fig. 3).
100 @0
Percent of insects
ol
Ss)
oO
e
on
| ea
oO OO Yo
1 3 5 7
Number of LD cycles
Fic. 3. Response curves of P. c-aureum two different
seasonal diphenisms to the number of long-day days,
where 0-day-old 4th-instar larvae from SD condi-
tions were exposed to LD cycles as given in the
abscissa and then transferred to SD regime. Open
and solid circles show the occurrence of summer
morph and vitellogenic female adults at 20°C.
Stage-dependent changes of the effects of LD-to-SD
transfer on the incidences of summer morph and
vitellogenic female adults
To examine how the effects of LD-to-SD trans-
fer on the incidences of summer morph and vitel-
logenic adult females change with the developmen-
tal stages, 0-day-old larvae of the 2nd, 3rd, 4th and
Sth instar were selected from a stock culture raised
from the egg stage under SD at 20°C. About
two-hundred and fifty larvae of the same stages
were grouped and subjected to a LD-to-SD trans-
fer at 20°C. The LD-to-SD transfer was carried
out at different/difinite developmental stages
which were changed depending on groups at one-
day intervals from day 2 of the first instar to day 2
of the Sth instar.
When larvae were subjected to a LD-to-SD
transfer before day 2 of the 2nd instar, all females
responded to SD and developed into autumn
morph adults, 13%-27% of which became vitel-
logenic. Percentages of females showing a re-
sponse to SD decreased gradually when the trans-
ferred stages were delayed from day 2 of the 2nd
instar. Fifty percent of the incidences of summer
morph and vitellogenic adults reached by a group
subjected to a LD-to-SD transfer on day 2 and day
0 of the 3rd instar, respectively. Critical stages of
LD-to-SD transfer after which P. c-aureum did not
show any response to SD were day 2 (for seasonal
morphs) and day 0 of the 4th instar (for reproduc-
tive activity) (Fig. 4).
100
76 Oe as
vy)
3)
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iz — ee —
= 504
G =— 5days —
©
oa
0 J e“d-0-0 Lye
Slimesl peleeant 0
10 i VO. a. keen
Stage of Pee transfer (LD to SD)
Fic. 4. Stage-dependent changes of the effects of LD-
to-SD transfer on the determination of P. c-aureum
two different seasonal diphenisms. Open and solid
circles show the occurrence of summer morph and
vitellogenic female adults at 20°C. “I0”, “II0”,
“TII0”, “IVO” and “VO” show the first days of the
first-, 2nd-, 3rd-, 4th- and Sth-instar larval stages,
respectively.
The results indicate that day 0 and day 2 of the
3rd instar seem to be the critical stages before
which P. c-aureum can experience sufficient num-
bers of LD days for achieving the 50% incidences
of summer morph and vetellogenic adults, respec-
tively.
Stage-dependent changes of the effects of SD-to-LD
transfer on the incidence of summer morph and
vitellogenic adult females
To assess how the effects of SD-to-LD transfer
on the incidences of summer morph and vitel-
Seasonal Diphenisms of Polygonia 729
logenic adults change with the developmental
stages, 0-day-old 4th- and Sth-instar larvae and
0-day-old pupae were selected from a stock culture
raised from the egg stage under SD at 20°C.
Two-hundred larvae and two-hundred pupae of
the same stages were grouped and were subjected
to a SD-to-LD transfer at 20°C. In this experi-
ment, the stage of the SD-to-LD transfer was
changed depending on the groups at 1-day inter-
vals from day 2 of the 4th instar to day 3 of pupa.
All females introduced to LD on days preceding
day 0 of the 4th instar developed into summer
morph adults. Percentage of the summer morphs
lowered quickly when the stage of the transfer was
delayed from day 0 of the 4th instar. Fifty percent
and one hundred percent of the incidences of the
summer morphs were achieved by groups intro-
duced to LD on day 3 and on day 5 of the Sth
instar, which corresponded to 3 days and one day
before larval-pupal ecdysis, respectively (Fig. 5).
With respect to the vitellogenic activity deter-
mination, P. c-aureum showed responses to a
SD-to-LD transfer different from those in morphs.
Seventy-four percent of females introduced to LD
on day 2 of pupae became vitellogenic. Critical
stage of the SD-to-LD transfer based on the 50%-
incidence of vitellogenic adults was found to lie on
day 2 of pupae, which is 5 days latter than that
obtained on the 50%-incidence of the summer
morphs (Fig. 5).
The results indicate that the developmental
stages at which P. c-aureum looses the photo-
100 5
un
U
o
wo
sg —4days
Oo 504
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wo
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o
- e
°
si \oret sie, he
TAP Tioger Vara 0 eae eee T ua T T T TT T T Teal:
1Y{0) vO PO Adult
Stage of photoperiodic transfer (SD to LD)
Fic. 5. Stage-dependent changes of the effects of SD-
to-LD transfer on the determination of P. c-aureum
two different seasonal diphenisms. Open and solid
circles show the occurrence of summer morph and
vitellogenic female adults at 20°C. “IV0”, “VO”,
“PO” and “adult” show the first days of 4th-instar
larval, Sth-instar larval, pupal and imagimal stages.
periodic responsiveness seems to vary depending
on the kinds of seasonal diphenisms of the insects.
DISCUSSION
Almost all adult females of autumn morphs in P.
c-aureum developed under complete short-day
photoperiods at 20°C entered reproductive di-
apause. Critical daylengths for occurrence of
autumn morph and reproductive diapause adult
females were about 13 hr, as has been reported for
morph determination in the same species [7].
The coincidental responses in the determination
of these two kinds of seasonal diphenisms dis-
appeared in the complete photoperiods at 25°C
(Fig. 1b). Differential fluctuations of the inci-
dences of these two kinds of seasonal diphenisms
were also observed in the larval groups subjected
to the asymmetrical skeleton photoperiods of 8L-
16D at 20°C (Fig. 2).
Physiological mechanism underlyint the photo-
periodic control of these two kinds of seasonal
diphenisms may involve clock and neuroendocrine
systems as had been demonstrated in many other
insects [13-18]. For the regulation of two kinds of
seasonal diphenisms, P. c-aureum may share clock
components functioning time-measurement of
photoperiod and accumulation of photoperiodic
information. This suppositon is based on almost
the same values of critical daylengths of these two
kinds of seasonal diphenisms which P. c-aureum
showed in the complete and asymmetrical skeleton
photoperiods, respectively (Figs. 1 and 2).
The differential responses of these two kinds of
seasonal diphenisms mentioned as above may de-
pend on differential responsiveness of neuroen-
docrine systems secreting summer-morph-produ-
cing hormone [12] and a factor activating corpora
allata in adults, which seems to do similar function
as allatotropin in Manduca sexta [19].
P. c-aureum required a LD-exposure of diffe-
rent length (five and four days) for achieving 50%
summer morph and vitellogenic adults (Fig. 3).
Photoperiod-sensitive stage in which P. c-aureum
shows the responsiveness to photoperiods seems to
begin at around the first larval ecdysis (Fig. 6) as
has been demonstrated in the determination of P.
xuthus pupal diapause [6]. This supposition is
730
Peaks of hemolymph
ecdysteroid titer
LD-to-SD
Critical stages transfer
for seasonal morph
determination
Photoperiod-sensitive stage
Critical stages for
reproductive diapause
determination
LD-to-SD
transfer
Photoperiod-sensitive stage
K. Enpbo, S. UENO et al.
SD-to-LD Brain
transfer NCCI+I
for seasonal morph determination
Brain SD-to-LD
ag transfer
for reproductive diapause determination
3rd
instar
First
instar
4th
instar
2nd
instar
Egg
Fia. 6.
5th
Pupa Adult
instar
Schematic representation of P. c-aureum photoperiod-sensitive stage with the critical stages of summer
morph and vitellogenic adult development. “NCC(I+II)” shows the nervi corporis cardiaci.
based on a hypothesis that groups of insects receiv-
ing a LD-exposure of 5 and 4 days before LD-to-
SD transfer should represent 50%-incidences of
summer morph and vitellogenic adult females. On
that day, P. c-aureum larvae undergo the first
larval ecdysis at 20°C (Fig. 4).
A similar explanation may be available for the
end of the photoperiod-sensitive stage; the stage
may come on day 1 of pupae, which is 5 days later
than the critical day of SD-to-LD transfer of the
incidences of summer morphs (Fig. 5). The stage
is one-day earlier than the critical days at which
summer-morph-producers no more require the
brain and the nervi corporis cardiaci which are
essential for summer morph and vitellogenic adult
development (Fig. 6) [10]. However, P. c-aureum
pupae are thought to have photoperiodic respon-
siveness on days following larval-pupal ecdysis.
This is based on the evidence that large numbers of
pupae introduced to LD on 6 days following
larval-pupal ecdysis showed a clear response and
developed into vitellogenic (autumn morph) adults
(Fig. 5). It may be that the a factor activating
corpora allata in P. c-aureum adults may be se-
creted in the late pupal stage according to the
photoperiodic imformation accumulated from day
0 of the 2nd instar to two or three days before
eclosion by the clock component (Fig. 6).
Here, we could not provide any evidence about
neuroendocrine system secreting a factor activat-
ing corpora allata (allatotropin?) in P. c-aureum
adults. But, it may become clear by further studies
on physiological mechanism underlying the photo-
periodic control of seasonal diphenisms of P. c-
aureum.
ACKNOWLEDGMENTS
This work was supported in part by Grant-in-Aid from
the Ministry of Education, Science and Culture, Japan
No. 03640606 to K.E.
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13
14
15
16
17
18
19
731
Neuroendocrine regulation of the development of
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ZOOLOGICAL SCIENCE 9: 733-739 (1992)
Scanning Electron Microscopy of Differentiating Chick
Ovaries during Embryonic Period
ATSUMI UKESHIMA
Kumamoto University, College of Medical Science,
Kumamoto 862, Japan
ABSTRACT— Asymmetrically developing right and left ovaries of the chick embryos from 2 to 19 days
of incubation were observed by scanning electron microscope (SEM). Surface epithelium of
undifferentiated gonad showed rough appearance with many vesicular protrusions and fillopodia, as
compared with the other regions of coelomic surface. After the ovarian differentiation started, surface
feature of the ovary was different between the right and left. In the left ovary, which made a normal
development, the surface was still rugged, and possessed many vesicular protrusions and fillopodia like
in undifferentiated period. In contrast, the right ovarian surface showed degenerating profile,
possessing few numbers of cytoplasmic protrusions. The most significant feature of the right ovary was
the fact that from 11 days onward, many holes were found on the surface. These holes exhibited a
variety in size and continued to inner medullary lacunae. Sometimes, a cell or cell aggregate which,
© 1992 Zoological Society of Japan
supposedly, were abandoned germ cells, were found at the opening of the holes.
INTRODUCTION
In the female chick embryo, the ovary develops
only on the left side, and the right degenerates to
atrophic remnant during an embryonic period.
This is due to the failure of the cortex formation in
the right gonad. Although primary sex cords are
equally formed on both sides during an early
differentiating period of the ovary, the secondary
cords, which is to form the cortex from 7 days of
incubation onward, do not appear on the right.
Eventurally the growth of the right ovary stops.
Although some ultrastructural studies on the
developing avian ovaries were present [1-4], they
described on the inner structure by transmission
electron microscopy (TEM). In the present study,
changes of the surface morphology of the right and
left ovaries which take the different pattern of
formation with the advance of development, were
compared by scanning electron microscopy
(SEM).
MATERIALS AND METHODS
Chick ovaries from stage 16 (staged by Hambur-
Accepted May 8, 1992
Received March 12, 1992
ger and Hamilton [5], about 2 days of incubation)
to 19 days were employed for this study. As the
growth of the right ovary stopped at the time of the
occurrence of secondary cords, female gonads
(Ovaries) were apparently different in size on the
right and left sides. Therefore, identification of the
gonadal sex was available.
Ovaries removed from the embryos were fixed
in 2.5% glutaraldehyde (phosphate buffer, pH
7.4) for 1.5 hr, and postfixed in 1% osmic acid
(phosphate buffer pH 7.4) for 1 hr. Some ovaries
were sectioned into thin pieces by free hand tech-
nique to observe the cut face. After dehydration,
materials were dried by critical point drying
method, coated with platinum-paradium, and
finally observed by JEOL scanning electron micro-
scope (Model JSM-6400F).
RESULTS
In the present study, a period of the ovarian
development is divided into two sections. The first
is an undifferentiated period. At the initiation of
this period (stage 16, about 2 days), gonadal
anlages are composed of columnar (thickened)
coelomic epithelium, or what is called the “germin-
al ridges”. Germ cells (primordial germ cells)
734 A. UKESHIMA
Fic. 1. Low magnification figure of the epithelial surface of undifferentiated gonad of a stage-16 embryo. Free
surface of the cells is swollen out and possesses many fillopodia, vesicles and short microvilli. 2,400.
Fic. 2. Mesonephric surface of a stage-16 embryo. As compared with the gonadal surface, few cytoplasmic processes
are seen, although cells are swollen out like in the gonadal surface. 2,400.
Fic. 3. Surface of the left ovary of a 8-day embryo. On the cell surface, numerous vesicular protrusions and some
fillopodia are present. 3,600.
Fic. 4. Right ovarian surface of a 8-day embryo. In contrast to the left ovary, the cell surface are flattened, and many
short microvilli are lined at the border of each cell. 2,400.
SEM of Embryonic Chick Ovaries 735
migrate into the germinal ridges via blood vessels _ epithelium is still thickned and pseudostratified,
from the extraembryonic sites [6-8]. and contains many immigrated germ cells. In the
The second is a differentiated period. At the right ovary, however, secondary cords are lacking.
beginning (7 days) of this period, secondary cords _‘The epithelium exhibits a thin, squamous structure
appear in the left ovary to form the cortex. The with no germ cells [4].
Fic. 5. Lower magnification figure of the left ovary of a 11-day embryo. The surface is very rugged, showing
depression and jutting out. 540.
Fic. 6. Left ovarian surface of a 16-day embryo. Cell surfaces are also covered with many vesicles, fillopodia as in
younger embryo. The cells in center retain the trace of cell division (arrow). The mitotic features according to
the ovarian growth were frequently observed, especially on the left ovary. 2,700.
@ E mg
Fic. 7. A cut surface of a 9-day left ovary. The thick surface epithelium (Ep) is composed of columnar cells. Note a
germ cell (asterisk) enclosed by the epithelial cells. 2,400.
Fic. 8. A sectioned view of a 9-day right ovary. Unlike the left ovary, the epithelium (Ep) is thin, and is composed of
squamous cells. Under the epithelium, lacunae of various sizes (asterisk) are frequently seen. 2,400.
736 A. UKESHIMA
Undifferentiated period
Surface of the germinal ridges (gonadal anlages)
as a whole showed a rough profile at low mag-
nification (Fig. 1). This was based on the presence
of vesicles of various sizes and fillopodia on the cell
surface. Free surface of each epithelial cell,
measuring 3.5 to 8 u~m in diameter, was swollen
out into the coelomic cavity. These surface fea-
tures of the undifferentiated gonads were
apparently different from those on the other re-
gions of the coelomic epithelium. For example,
epithelial surface of mesonephros was smooth ow-
ing to poor protrusions, although each cell was
swollen out like in the gonadal surface (Fig. 2).
Fic. 9.
Whole view of the right ovary from a 16-day embryo. Many holes are found on the ovarian surface. Note a
posterior end (arrows) of the ovary (Ov) incorporated to the mesonephros (Ms). X90.
Fic. 10.
oval shape. 250.
Surface of a 19-day right ovary. Holes of various sizes are seen. The opening of the holes is usually round or
SEM of Embryonic Chick Ovaries 737
Differentiated period
From 7 days onward, which ocrresponded to the
beginning of the secondary cord formation, surface
of the developing left ovary still showed similar
profile as in the undifferentiated gonads; each cell
surface was usually swollen out, possessing vesicu-
lar protrusions of various sizes (Fig. 3). Among
those, long fiber-like structures were seen extend-
ing from the edge of the cells. The free surface of
the cell was 3 to 6 ~m in diameter, usually about 4
ym.
On the other hand, the mght ovary, which
ceased to make further development because of
the failure of the secondary cord formation, exhi-
bited a smooth and flattened surface (Fig. 4). The
free surface of each cell was somewhat larger than
that in the left ovary, measuring 4.6 to 14 wm in
diameter. At the margin of each cell surface, short
microvilli or protrusions were seen.
The surface of the left ovary became more
prominent with developmental stages, showing
irregularly rugged features at lower magnification
(Fig. 5). Some parts were protruded, the others
hollowed. Small hollows were often observed.
Higher magnification revealed the rough surface of
the epithelial cells covered by many vesicular
protrusions. Some cells were elongated and con-
Fic. 11.
nected with other cells by the intercellular bridge,
showing the vestige of cell division (Fig. 6).
The cut surface revealed difference in the
epithelial composition of both ovaries. The epithe-
lium of the left ovary was thickned like in earlier
stages. Germ cells were frequently seen to be
involved among these columniated epithelial cells
(Fig. 7). In the right ovary, however, the epithe-
lium was thin and composed of the squamous cells
(Fig. 8), possessing no germ cells. Just under the
epithelium, medullary lacunae were often obser-
ved.
The most prominent feature of the right ovary
was the holes on the surface, which appeared from
11 days onward (Figs. 9 and 10). Although 11-day
ovary contained several holes on the surface, they
increased in number with the advance of develop-
ment. In 17 days, almost whole surface of the right
ovary were occupied with the holes. The holes
varied in size, measuring 12 to 47 um in diameter.
The right ovary in advanced stage of embryo was
seen to be incorporated partly to the mesonephros,
the border partly being obscured (Fig. 9). The
epithelial surface was flat and scanty of microvilli.
In the surroundings of the holes, however, the
cytoplasmic protrusions such as fillopodia and mic-
rovilli were seen increasing in number (Fig. 11).
The cut surface of right ovary revealed the holes
ie : j eee (12)
A high magnification figure of a hole of the right ovary from a 13-day embryo. Around the hole many
microvilli or long fillopodia are seen, although superficial epithelial cells have few microvilli. 1,800.
Fic. 12. A sectioned view of the right ovary from a 19-day embryo. This figure shows that the hole on the ovarian
surface continues with the medullary lacunae (La). Arrow: pathway of the hole to the lacunae. x700.
738 A. UKESHIMA
Fic. 13.
This figure shows a cell aggregate coming out of the opening of a hole on the right ovary from 19-day
embryo. The aggregate is composed of several adhered cells. The arrows show the adhered portions of the cells.
x3.600.
continuing to the medullary lacunae (Fig. 12),
while the hollows found on the left ovarian surface
were not continuous to the inner structure.
At the opening of the holes, free cells were
occasionally found after 16 days of incubation.
These cells were observed as a single cell, or a cell
aggregate in which several cells adhered to each
other (Fig. 13). The number of the cells were not
alterable with increasing embryo age.
DISCUSSION
In the chick embryo, the ovaries were different
in size between the right and left sides. This is due
to the failure of development of the right ovary.
The surface morphology of the asymmetrically
developing ovaries were first revealed by SEM in
the present study.
On the surface of the right atrophic ovaries,
many holes were present at later stage of develop-
ment and continued to inner medullary lacunae.
Although it is not clear what mechanism exists
about this continuity in the medullary lacunae of
the atrophic right ovary, it might be concerned
with the degeneration of germ cells from under-
standing in our previous study [4], which described
on germ cells released in the medullary lacunae of
degenerating right ovaries.
In some cases, a cell or cell aggregate were
found at the opening of the holes. Also judging
from our previous study [4], the cells found at the
opening may be exhibiting a terminal phase of
those germ cells. More accurate identification of
these cells, however, should be done by the histo-
logical technique.
As to birds, microscopic studies on the lacunar
system of the normal left ovary have been per-
formed by Callebaut er al. [9, 10]. They indicated
that the lacunar system continued to the coelomic
(peritoneal) cavity at the dorsal (proximal) side of
the left ovary. In the present study, SEM observa-
tion have been made only on the surface of both
ovaries, and not on the dorsal dide, demonstrating
that no holes were present on the left ovarian
surface.
The cell aggregates found at the opening of the
holes in the right ovary were often adhered tightly
to each other. This aggregate is, presumably, a
mass made of germ cells and lacunar epithelial
cells. Similar structures were found in the right
medullary lacunae in our previous study [4].
At the beginning of ovarian differentiation,
SEM of Embryonic Chick Ovaries
germ cells were observed in the epithelium of the
left ovary, but never in that of the right. Initially,
migrating germ cells were equally existed in the
columnar epithelium of both gonads. Thereafter,
almost all germ cells in the right ovary have moved
to the medulla with a part of the epithelium, while
epithelial cells have changed to a squamous shape.
Therefore, germ cells in the right ovary were
localized in the medulla, but none in the epithe-
hum.
At the cut surface of the ovary, the germ cells
within a epithelium were easily identified, because
the germ cells in the epithelium were round in
shape and larger than the mitotic epithelial cells [7,
Sh, Lil],
As secondary cords are formed into a cortex
only on the left side, the left ovary developed
larger in size than the right. At 7 days of incuba-
tion, however, difference in ovarian size between
the right and left sides was imperceptible, because
it was just after initiation of the cortex formation.
Therefore, materials from 7-day embryo were not
employed.
REFERENCES
1 Narbaitz, R. and Adler, A. (1966) Submicroscopic-
al observations on the differentiation of chick
gonads. J. Embryol. Exp. Morphol., 16: 41-47.
2 Yamada, K. and Amanuma, A. (1980) Fine struc-
ture of Balbiani body in germ cells of the chick
embryo. J. Predental Fac. Gifu Coll. Dentistry, No.
10
739
6: 87-103. (in japanese).
Yamada, K. and Amanuma, A. (1985) Ultrastruc-
ture of interstitial cells in the gonad of quail embryo.
J. Liberal Arts Asahi Univ., No. 11: 113-125.
Ukeshima, A. and Fujimoto, T. (1991) A fine
morphological study of germ cells in asymmetrically
developing right and left ovaries of the chick. Anat.
Rec., 230: 378-386.
Hamburger, V. and Hamilton, H. L. (1951) A
series of normal stages in the development of the
chick embryo. J. Morphol., 88: 49-92.
Fujimoto, T., Ukeshima, A. and Kiyofuyi, R. (1976)
The origin, migration and morphology of the pri-
mordial germ cells in the chick embryo. Anat. Rec.,
185: 139-154.
Ukeshima, A. and Fyimoto, T. (1984) Ultrastruc-
ture of primordial germ cells in the early chick
embryo. In: Ultrastructure of Reproduction. (J.
Van Blerkom and P. M. Motta eds.) Martinus
Nijhoff Pub., Boston, 12-18 pp.
Ukeshima, A., Yoshinaga, K. and Fujimoto, T.
(1991) Scanning and transmission electron micros-
copic observations of chick primordial germ cells
with special references to the extravasation in their
migration course. J. Electron Microsc., 40: 124-128.
Callebaut, M. (1979) The avian ovary is an open
organ.; A study of the lacunar system. Anat.
Embryol., 158: 103-119.
Callebaut, M., Meeussen, C. and Nassauw, L. V.
(1988) The early development of the lacunar system
in the avian ovary. Med. Sci. Res., 16: 1131-1133.
Ukeshima, A. and Fujimoto, T. (1978) Scanning
electron microscopy of the primordial germ cells in
early chick embryos. J. Electron Microsc., 27: 19-
24.
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ZOOLOGICAL SCIENCE 9: 741-747 (1992)
© 1992 Zoological Society of Japan
The Acid-Insoluble Organic Matrix of Spicules in the Sea
Urchin Hemicentrotus pulcherrimus
SHOGO NAKAMURA, RYOKO KAGOTANI, HIROE FUJISAKI
and MANABU K. KoJIMA
Department of Biology, Faculty of Science,
Toyama University, Toyama 930, Japan
ABSTRACT— The structure of the acid-insoluble organic matrix (ATOM) of the spicules of sea urchin
larvae was examined by light microscopy and transmission electron microscopy. The spicules were
isolated from pluteus larvae of Hemicentrotus pulcherrimus, and AJOM was obtained by demineraliza-
tion of the spicules with 0.1 N acetic acid. We found that AIOM has many fine fibers (diameter: 16-27
nm) and two types of granules.
INTRODUCTION
A mineralized skeleton of spicules is one of the
most conspicuous structures in sea urchin
embryos. Previous investigations of the develop-
ment of the skeleton in sea urchin larvae showed
that the primary mesenchyme plays a major role in
spicule formation [1-4]. However, the control of
spicule formation has not yet been fully resolved.
Calcite, CaCO3, and small amount of MgCO; are
the inorgaic components of the spicules [5]. Oka-
zaki [1] showed that spicules contain an organic
matrix which was insoluble after acid-treatment.
Benson ef al. [6] revealed that EDTA- or acid-
insoluble organic matrix contains fibrous lamella.
Although acid- or EDTA-soluble organic matrix
had several glycoproteins and proteins [7, 8], the
components of acid- or EDTA-insoluble matrix
are not known. To obtain further information
about biomineralization in the sea urchin embryo,
we have observed the structure of the acid-
insoluble organic matrix (AIOM) by light and
electron microscopy. We found that AIOM con-
tains many fibers and two types of granules.
Accepted May 28, 1992
Received March 9, 1992
MATERIALS AND METHODS
Gametes Hemicentrotus pulcherrimus were
collected from the coast of Toyama Bay and were
maintained at 14°C in aquaria with circulating sea
water. Gametes were obtained by injecting 0.5 M
KCl into the body cavity. Sperm were collected
directly from the coelomic cavity and stored “dry”
in the refrigerator. Eggs were collected and
washed 2 times in filtered natural sea water
(FNSW).
Isolation of organic matrix Fertilized eggs were
washed in FNSW and cultured to plutei at 18°C
with constant stirring (~1lround/sec). Larvae
were collected by centrifugation (at 3,000 g for 10
min) and washed once in cold (4°C) Ca*t- and
Mg**-free sea water (CMFSW: NaCl 26.5 g, KCI
0.7 g, pH was adjusted to 8.2 by the addition of 0.5
M NaHCoO;) [9]. They were resuspended in
CMFSW, shaken vigorously, then centrifuged at
1,000 g for 5 min. The pellet was resuspended in
10 vol of 10 mM Tris buffer (pH 7.5) which con-
tained 0.5 mM phenylmethylsulfonyl fluoride, and
pipetted vigorously. The suspension was centri-
fuged at 1,000 g for 5 min. The pellet was resus-
pended in 10 vol of 2% TritonX-100, 4% sodium
deoxycholate, 20mM Tris buffer (pH7.4) and
pipetted strongly, then centrifuged at 1,000 g for 5
min. This procedure was repeated 2-3 times to
742 S. Nakamura, R. KAGOTANI et al.
obtain crude fraction of spicules as the pellet. The
crude fraction was resuspended in 5vol of the
same Tris buffer containing detergents and 5 vol of
5% sodium hypochlorite in 20 mM Tris buffer (pH
7.5). The suspension was pipetted and centrifuged
at 25 g for 3 min. The pellet was treated with 5%
sodium hypochlorite for 10min and washed 3
times with artificial sea water (ASW: NaCl 26.5 g,
MgSO,:7H20 11.9g, KCl 0.7 g, CaCl 1.2 g, pH
was adjusted to 8.2 by the addition of 0.5M
NaHCOs) [9].
Micrographs of isolated spicules were taken by
drak-field microscopy (Olypus BH2 microscope
with a BH2-DCW dark-field condenser).
Electron microscopy Isolated spicules were de-
mineralized by resuspending them in equal vol of
0.1 N acetic acid and incubating for 30 min at 0-
4°C. The suspension was spun at 16,000 g for 10
min, and the acid-insoluble organic matrix
(AIOM) was obtained as the pellet. The AIOM
was fixed in 2% glutaraldehyde in 80% ASW for 1
hr at room temperature, and then fixed again in
1% glutaraldehyde in 0.1M sodium cacodylate
(pH 7.0) for 1 hr. The samples were post-fixed in
1% OsOy, in 0.1 M sodium cacodylate (pH 7.2) for
1 hr. After rinsing 0.1 M sodium cacodylate buffer
(pH 7.2), the samples were dehydrated through a
ethanol series. They were infiltrated and embed-
ded in epoxy resin (Epok 812, Oken Shoji Co.,
Ltd., Tokyo). Sections were cut with glass knives
on a Sorvall MT1 Porter-Blum ultramicrotome,
and stained with uranyl acetate and Reynold’s lead
citrate. To survey the purity of isolaed spicule,
whole mounts were prepared. Ultrathin sections
and whole mounts were examined with a JEOL
100SX electron microscope operated at 80 kV.
RESULTS
We isolated many spicules in almost intact form
from pluteus larvae of Hemicentrotus pulcherrimus
(Fig. 1b). There was very little contamination in
the fraction of isolated spicules (Fig. 1b) and the
surface of the spicules was clean (Fig. 2). When
the isolated spicules were treated with 0.1 N acetic
acid for 30 min, demineralization occurred and
thread-like structures remained (Fig. lc), as re-
Fic. 1.
sea urchin, Hemicentrotus pulcherrimus.
Preparation of AIOM from pluteus larvae of the
Pluteus
larvae (a: 72 hr after the insemination) were used for
isolation of spicules which appeared free of con-
tamination by dark-field microscopy (b). After
isolated spicules were demineralized with 0.1 N ace-
tic acid, thread-like structures with granular varico-
sities remained (c). Bar, 100 «~m.
ported by Okazaki [1].
granules on the thread-like structures.
There were many fine
Those
granules were never seen in the fraction of isolated
spicules. We call this thread-like structure the
acid-insoluble organic matrix (AIOM). AIOM
was examined with transmission electron micros-
copy. Figure 3 shows that AIOM has many fibers
Organic Matrix of Spicules 743
which are twined loosely (diameter: ~27 nm) with
a few filaments (Fig. 4a) or tightly (diameter: ~ 16
nm) (Fig. 4b). We could not see a periodic pattern
on the fibers. The fibers ran parallel with each
other in some regions and they made an eddy
pattern in other regions.
Moreover, AIOM contained two types of gran-
ules (Fig. 3). Granules whose appearances re-
minded us of a cell nucleus (N-granules) lacked
limiting membranes (Figs. 3, 5a). Many fine
fibrous structures were present in the electron
Fic. 2. Whole mount EM of ioslated spicules. No translucent compartment of N-granules (Fig. 5a).
contamination is evident around the spicule surface. | We often observed disintegrating N-granules with
Bar, 5 wm. discharged fibrous structures (Fig. 5a) which
looked like filaments in the loosely twined fibers
(Fig. 4a). The other type of granules (V-granules)
Fic. 3. A typical ultrathin section of AIOM. Many fine fibers and two types of granules were found in this specimen.
The fibers run parallel in some regions, and in eddy patterns in other regions. N: N-granule. V: V-granule. Bar,
2 pm.
Fic. 4. High magnification TEM views of the fibers in AIOM. The fibers are twined loosely with a few filaments (a)
or more tightly (b). Bar, 0.2 ~m.
Fic. 5. High magnification views of two types of granules in AIOM. The nucleus-like granule (N-granule) (a) has no
clear membrane and many fine fibers in the electron translucent parts. This N-granule was disintegrating with
discharged filaments (arrows). The other type of granules (V-granules) have vacuoles surrounded with electron
dense deposite (b). Bar, 0.5 um.
744 S. Nakamura, R. KaGorani et al.
Organic Matrix of Spicules 745
746 S. NAKAMURA, R. KAGOTANI et al.
showed homogenous and low electron density, and
had large vacuoles with peripheral deposits of
electron dense materials in it (Figs.3, 5b). V-
granules had no outer membrane and took various
shapes. They were seen more frequently than
N-granules. Preliminary observations show that
N-granules and V-granules have various diameters
(N-granules: 2.5 ~m-—3.2 um; V-granules: 1.4 ~m—
2.4 wm).
DISCUSSION
We have found that spicules of sea urchin
embryos contain many fine fibers and two types of
granules. Our preliminary observations show that
the same types of fibers and granules that we
observed in Hemicentrotus pulcherrimus embryos
exist in spicules of Pseudocentrotus depressus
embryos (data not shown). Benson ef al. [6]
reported that the organic matrix of spicules had
fibrous lamellae. However, they did not show the
fine structure of the fibrous lamella and did not
report the existence of granules.
In the blastocoel of the sea urchin embryo, there
is an abundunce of fibrillar matrix which may
consist of collagen [10]. However, we believe that
fibers and granules in our preparation are not
contaminants of blastocoelic fibrillar matrices or
substances, since (1) no contaminants were evident
around the surface of isolated spicules by light and
electron microsopy (Figs. 1b, 2), (2) we observed
only one type of fiber and two types of granules in
AIOM. Although the diameter of the fibers varied
from 16 nm to 27 nm, we think they are made from
the same material(s) and that the difference in
diameter reflects their formation or assembly.
It is well known that vertebrate bone predomi-
nantly contains tvpe I collagen fiber. Pucci-
Minafra et al. [11] have reported that the spicule of
a sea urchin embryo contains collagen and which
may play a role in the formation of the organic
matrix of the spicules. Several investigators re-
ported that collagen metabolism is required for
spicule formation [12-14]. On the other hand,
Benson et al. failed to detect the presence of
hydroxyproline-rich collagen by structural [6] and
biochemical studies [7], and they claimed that
spicules isolated by the methods of Pucci-Minafra
et al. were impure. We think the fibers seen in
AIOM are probably not collagen since they did not
show periodic banding, and the proteins did not
have typical mobilities of collagen in SDS-PAGE
(preliminary data not shown).
We suppose the fibers give strength and flexibil-
ity to the spicule and the granules are related to the
fiber formation. Whole AIOM may have an
important role in the formation of the spicule.
Biochemical studies are under way to define their
natures and functions.
ACKNOWLEDGMENTS
We thank Drs. S. L. Tamm and S. Pimple (BUMP,
MBL, Woods Hole) for their critical readings of this
manuscript.
REFERENCES
1 Okazaki, K. (1960) Skeleton formation of sea
urchin larvae. II. Organic matrix of the spicule.
Embryologia, 5: 283-320.
2 Wolpert, L. and Gustafson, T. (1961) Studies on the
cellular basis of morphogenesis of the sea urchin
embryo. Development of the skeletal pattern. Exp.
Cell Res., 25: 311-325.
3 Okazaki, K. (1965) Skeleton formation of sea
urchin larvae. V. Continuous observation of the
process of matrix formation. Exp. Cell Res., 40:
585-596.
4 Okazaki, K. (1975) Spicule formation by isolated
micromeres of the sea urchin embryo. Amer. Zool.,
15: 567-581.
5 Okazaki, K. and Inoué, S. (1976) Crystal property
of the larval sea urchin specicule. Develop., Growth
and Differ., 18: 413-434.
6 Benson, S., Jones, E. M. E., Crise-Benson, N. and
Wilt, F. (1983). Morphology of the organic matrix of
the spicule of the sea urchin larva. Exp. Cell Res.,
148: 249-253.
7 Benson, S. C., Benson, N. C. and Wilt, F. (1986)
The organic matrix of the skeletal spicule of sea
urchin embryos. J. Cell Biol., 102: 1878-1886.
8 Venkatesan, M. and Simpson, R. T. (1986) Isola-
tion and characterization of spicule proteins from
Strongylocentrotus purpuratus. Exp. Cell Res., 166:
259-264.
9 Okazaki, K. (1956) Skeleton formation of sea
urchin larvae. I. Effect of Ca concentration of the
medium. Biol. Bull., 110: 320-333.
10 Crise-Benson, N. and Benson, S. C. (1979) Ultra-
structure of collagen in sea urchin embryos. Wilhelm
11
12
Organic Matrix of Spicules
Roux’s Arch Dev. Biol., 186: 65-70.
Pucci-Minafra, I., Casano, C. and La Rosa, C.
(1972) Collagen synthesis and spicule formation in
sea urchin embryos. Cell Differ., 1: 157-16513.
Golob, R., Chetsanga, C. J. and Doty, P. (1974)
The onset of collagen synthesis in sea urchin
embryos. Biochim. Biophys. Acta, 349: 135-141.
13
14
747
Gould, D. and Benson, S. (1978) Selective inhibi-
tion of collagen synthesis in sea urchin embryos by a
low concentration of actinomycin D. Exp. Cell Res.,
112: 73-78.
Blankenship, J. and Benson, S. (1984) Collagen
metabolism and spicule formation in sea urchin
micromeres. Exp. Cell Res., 152: 98-104.
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ZOOLOGICAL SCIENCE 9: 749-755 (1992)
© 1992 Zoological Society of Japan
Scleral Fibroblasts of the Chick Embryo Can Proliferate
without Transferrin in Protein-Free Culture
YosHie Ouya!, KAzUuo WATANABE!”*, NoBuo SHIMAMOTO2
and Minoru Amano!
'Cell and Developmental Biology Laboratory, Faculty of Integrated
Arts and Sciences, Hiroshima University, Hiroshima 730, and
2DNA Research Center, National Institute of
Genetics, Mishima 411, Japan
ABSTRACT—Scleral fibroblasts of the chick embryo can proliferate in vitro without transferrin. We
prepared a monoclonal antibody, TFY004, which was reactive against avian-transferrin, and checked
the possibility of an autocrine secretion of transferrin in the cells.
Dot-blot immunoassay of the
conditioned medium by the use of TFY004 indicated that the conditioned medium did not contain
detectable amount of transferrin. We tested muscle trophic activities of the conditioned medium to
myogenic cells to detect transferrin activities in the conditioned medium. The results also indicated no
presence of transferrin in the conditioned medium. Furthermore, the supplementation of transferrin
had no effect for survival and proliferation of the scleral fibroblasts. All these results suggested that, in
the scleral fibroblast culture, mechanisms different from the external transferrin-mediated one must be
involved to uptake iron from medium into cells.
INTRODUCTION
Living cells inevitably requie iron. It is generally
accepted that the cells utilize environmental iron
by an efficient transport system with the use of
transferrin, a soluble protein with high iron-
binding capacity [1, 2]. One of the reason for
adding serum in the cell cultures is to supply a
sufficient amount of serum-transferrin. The trans-
ferrin binds efficiently irons in the culture medium,
and the iron-bound transferrin is internalized by
endocytosis [3] after specific binding to the cellular
receptor [4]. In consequence, the supplementation
of high concentration of transferrin, 5-10 “g/ml,
is a general format for serum-free cultures [5].
Interestingly, the scleral fibroblasts of chick
embryo do not require supplementation of trans-
ferrin to the protein-free culture medium for their
proliferation [6]. Then, a question arises, how the
Accepted May 28, 1992
Received May 6, 1992
3 To whom all correspondence should be addressed.
scleral fibroblasts uptake environmental iron,
which is necessary for cell survival.
One possible mechanism in an autocrine secre-
tion of transferrin by the cells themselves. In fact,
we reported that the scleral fibroblasts proliferated
by producing multiple autocrine growth factors
into the conditioned medium [6, 7]. Dittmann et
al. reported that HL-60 cells produced a transfer-
rin-like protein as an autocrine factor, which
reacted with anti-transferrin antibody [8], and also
Kohgo et al., reported K562 cells could proliferate
without transferrin by secreting immuno-reactive
transferrin as an autocrine factor [9].
In this study, we tried to detect transferrin in the
conditioned medium of the scleral fibroblast cul-
ture.
MATERIALS AND METHODS
Chick embryos were obtained by incubating
eggs of randomly bred Japanese fowl purchased
from Yamagishism Association, Japan.
750 Y. Onya, K. WATANABE et al.
1. Cell culture
Scleral fibroblasts
Procedures for culturing scleral fibroblasts were
described previously [6, 7]. In brief, the scleral
fibroblast layers were isolated from the eyeballs of
chick embryos (stage 38 or stage 39) [10], and
incubated with 2 ml of 0.2% collagenase at 37°C
for 40 min, and flushed gently in 2 ml of EF-
medium (1:1 mixture of Dulbecco’s modified
Eagle medium and Ham’s F-12 medium), using a
Pasteur pipette to dissociate tissues into single
cells. The dissociated cells were inoculated at an
initial density of 5 x 10° cells in 100-mm plastic dish
with 8ml of EF-medium and cultured in 5%
CO,-95% moist air at 38°C.
Myogenic cells
Muscle tissues were isolated from the femoral
region of stage 38 embryos. Procedures for dis-
sociation were the same as in the case of scleral
fibroblasts. The cells were inoculated at an initial
density of 5X10? cells in 35-mm plastic dish (Fal-
con 3001; Becton Dickinson Co., U.S.A.) with 2
ml of EF-medium supplemented with 10% horse
serum (GIBCO, U.S.A.) and were incubated in
humidified 5% CO,-incubator at 38°C.
2. Preparation of conditioned medium and SAF-I
from the scleral fibroblast culture
After the first medium change on the third day
of culture, the conditioned media were harvested
every second day, i.e., the fifth, seventh and
nineth days of culture. Cell densities were 120
cells/mm? at the third day and 650 cells/mm” at
the nineth day.
The procedures for preparing the SAF-I (scleral
autocrine factor-I) and its characterization were
described previously [6]. In brief, pooled con-
ditioned medium was concentrated by the use of
ultrafiltration, removing molecules less than 10
kDa. The concentrated conditioned medium was
applied to DEAE-Sepharose column chroma-
tography at pH 8.0, and proteins were eluted with
0-0.35 M NaCl gradient. We called a fraction,
eluted at about 0.2 M and showed a heat-stable
growth-promoting activity, as SAF-I.
3. Preparation of ovotransferrin as an avian trans-
ferrin
Ovotransferrin was extracted from chick egg
white after Kimura’s methods [11]. In brief,
acetone powder of egg white was prepared and
dissolved in FeCl; solution. The solution was
subjected to preparative DEAE-Sepharose, and
next to CM-Sepharose column chromatography.
A fraction which showed a single peak (absorb-
ance 280 nm), manifested also a clear single band
with molecular weight of ca. 80kDa by SDS
polyacrylamide-gel electrophoresis (SDS-PAGE)
[12]. We call the fraction as purified ovotrans-
ferrin.
4. Preparation of monoclonal antibody against
transferrin
As an antigen, 0.8 mg of the purified ovotrans-
ferrin was dissolved in 0.25 ml of distilled water,
mixed with the same volume of complete Freund’s
adjuvant and injected intraperitoneally to a
BALB/c mouse. Twelve days after the primary
injection, booster injection was made with an
adjuvant-free antigen. Three days later, spleen
cells were harvested and fused with myeloma cells
(Sp2/0-Ag-14) [13] by the use of polyethylenegly-
co16000 (Koch-Light Co., Germany) as described
by Galfre et al., [14]. HAT-selected hybridoma
cells were cloned by limiting dilution and the
supernatant media were screened by ELISA
method using horse-radish peroxidase (HRP)-
conjugated goat anti-mouse immunoglobulins
(ZYMED LAB. Inc., U.S.A.).
We obtained 15 hybridoma clones producing
antibodies against transferrin. But unfortunately,
most positive clones attenuated rapidly, i.e., they
ceased to produce antibodies, or stopped to pro-
liferate and died. Finally a hybridoma clone,
TFY004 clone, was remained. We recloned the
TFY004 cells, which left sufficient amount of hy-
bridoma supernatant (TFY004), which specifically
reacted with purified ovotransferrin on nitrocellu-
lose filter (Fig. 1).
5. Dot-blot immunoassay
Every one ml of the solutions containing various
concentration of the test samples was blotted to
Cell Proliferation without Transferrin 751
ovalbumin
BSA
va
—
Ing
300ng
eS 100ng
30ng
10ng
3ng
Fic. 1. Dot-blot immunoassay of purified ovotransfer-
rin, ovalbumin and BSA (bovine serum albumin)
with hybridoma supernatant, TFY004. Each 1 ml of
serially-diluted ovalbumin (No. 300-00711; Wako,
Japan), BSA (No. 017-12961; Wako) or purified
ovotransferrin (Tf) were blotted to a nitrocellulose
filter and reacted with TFY004. The concentrations
were indicated at the right.
nitrocellulose filter (No. 162-0115; BIO-RAD,
U.S.A.) by the use of Milliblot (Millipore Ltd.,
U.S.A.). The filter was washed with Ca**- and
Mg**-free phosphate buffered saline (PBS) and
reacted with non-diluted hybridoma supernatant,
TFY004, for 1hr. Then, 60-times dilution of
HRP-labelled goat anti-mouse immunoglobulins
(ZYMED LAB., Inc.) was added and visualized
by diaminobenzidine. All procedures were done at
room temperature and filters were washed with
PBS containing 0.05% Tween-20. Non-specific
binding of antibodies was blocked with PBS con-
taining 3% of commercial skim milk.
6. Measurement of growth-promoting activity
Growth-promoting activity was assayed as an
enhancement in *H-thymidine incorporation into
acid-insoluble fraction of the cells. Detailed condi-
tions for culturing and labelling were given pre-
viously [6, 7] and also in figure legends for Figure 5
and 6. After labelling, the cell monolayer was
lysed with 0.1 N NaOH and lysate was poured
onto a glass microfiber filter after Kawahara’s
methods [15]. The filter was washed three times
with 5% TCA and once with absolute ethanol.
Radioactivity was counted with an Aloka LSC-
7000 liquid scintillation counter [15].
RESULTS
Conditioned medium did not contain transferrin
according to dot-blot immunoassay
SDS-PAGE of the conditioned medium indi-
cated the presence of a protein with molecular
weight near 80 kDa, which was similar to transfer-
Its concentration in the medium was esti-
We purified
rins.
mated to be 150 ng/ml (Fig. 2).
conditioned medium with DEAE-sepharose chro-
CM
SAF-|
«— 80kDa—,
aay
hae sae
320° >
Fic. 2. SDS-PAGE of the conditioned medium (CM)
and the partially purified growth promoting factor
(SAF-I) separated from the conditioned medium.
Ten micro litter of the acid-insoluble materials of the
conditioned medium was applied, corresponding to
0.1 ml of conditioned medium. In the case of SAF-I,
10 wl of the solution containing 4 ug of protein was
applied.
752 Y. Onya, K. WATANABE et al.
matography. The partially purified fraction named
SAF-I [6], also showed a band of ca. 80kDa
protein (Fig. 2).
However, there found no substance to react with
anti-transferrin antibody in the conditioned
medium by dot-blot immunoassay. If transferrin
existed, it should be less than 1 ng/ml in the
conditioned medium (Figs. 1, 3).
SDS-Tf
tml
300,
30 pI
10 pl
Sp! 1ng
tpl 0.1ng
Fic. 3. Dot-blot immunoassay of the conditioned
medium of scleral fibroblasts with hybridoma super-
natant, TFY004. Each 1 ml of serially-diluted con-
ditioned medium were blotted to nitrocellulose
filter. The volume of conditioned medium used was
indicated at the left. As the controls, each | ml of
ovotransferrin and SDS-treated one (SDS-Tf) were
blotted to the same filter and reacted with TFY004.
Their concentrations were indicated at the right.
Conditioned medium had no effect on myogenic
cells
Addition of 3 “g/ml of purified ovotransferrin
to myogenic cells brought about prominent muscle
trophic effects (Fig. 4), as reported by Ozawa’s
group [2, 16]. On the other hand, 50-fold concen-
trated conditioned medium of the scleral fibro-
blasts did not promote the proliferation and dif-
ferentiation of myogenic cells (Fig. 4).
Consistently, the conditioned medium and SAF-
I, which showed DNA-synthesis promoting activ-
ity to the scleral fibroblasts, did not promote any
DNA synthesis of the myogenic cells, under the
condition that the supplementation of ovotransfer-
rin specifically promoted it (Fig. 5).
The addition of ovotransferrin had no effect on the
growth of scleral fibroblasts
The addition of 6 4g/ml of ovotransferrin, a
routine concentration in serum-free cultures, had
no DNA-synthesis promoting effect to the scleral
fibroblasts, although insulin had prominent effect
to promote DNA synthesis (Fig. 6).
Increasing the concentration of ovotransferrin
from 6 ng/ml to 50 ug/ml, we obtained the same
results (data not shown).
DISCUSSION
The scleral fibroblasts can rapidly proliferate in
vitro under protein-free medium [6]. We tested a
postulation that the scleral fibroblasts secreted
transferrin into conditioned medium as an auto-
crine factor.
It is known that the biological activities of
transferrins manifest class-specificity among verte-
brate animals [2, 17], i.e., mammalian-type trans-
ferrin does not manifest its biological activity to
avian cells, and vice versa. So, we purified ovo-
transferrin from chick egg white as an avian-type
transferrin [11, 17] and used it for positive control
experiments in the biological assays (Figs. 4, 5, 6).
At the same time, we prepared an specific mono-
clonal antibody, TFY004, against the ovotransfer-
rin. Since the TFY004 could not recognize SDS-
treated ovotransferrin (Fig. 3), the epitope of
TFY004 seemed to be protein conformation, not
to be carbohydrate moiety.
The obtained results indicated; (1) the con-
ditioned medium did not contain immunochemi-
cally-detectable amount of transferrin. The dot-
blot analysis indicated that it contained, if existed,
less than 1 ng/ml (Figs. 1, 3), much less than the
effective concentration, 800 ng/ml [18], of trans-
ferrin on growth of myogenic cells. (2) The
Cell Proliferation without Transferrin 753
conditioned medium did not mainfest any muscle
trophic effects (Fig. 4) or growth-promoting effect
(Fig. 5) upon myogenic cells even with the use of
concentrated one, which estimated to contain ca.
7-10 ug/ml of the 80 kDa protein. These results
indicated that there are no significant amount of
transferrin in the conditioned medium, and lead us
to discard the possibility of autocrine secretion of
transferrin in the scleral fibroblasts. The protein
with molecular weight of ca. 80 kDa in the con-
ditioned medium was considered not to be trans-
ferrin.
100 hm
Fic. 4. Myogenic cell cultures treated with ovotransferrin (3 ~g/ml) or conditioned medium (final concentration was
50-fold concentrated from original conditioned medium), showing prominent differences in growth and
differentiation on the 4th day after inoculation. For culture conditions, see Materials and Methods.
Then, how do the scleral fibroblasts satisfy iron
requirement for their survival and proliferation?
Interestingly, the supplementation of 6 ng—S0 ug/
ml of ovotransferrin showed no effects on their
proliferation. This fact seems to suggest the scleral
fibroblasts lack transferrin receptor, since it is
generally accepted that the transferrin receptors
increase in the rapidly proliferating cells [2]. In
another word, the fact implicates the uptake of
iron without participation of soluble transferrin in
the scleral fibroblasts.
One possible mechanism is the scleral fibroblasts
=)
Nn
ms
3H-Thymidine incorporation (cpm x 10-3)
3H-Thymidine incorporation (cpm x 10-2)
=
(o)
,
]
Y. Onya, K. WATANABE et al.
BSA
=
oO
Tf
Fic. 5.
SAF-|
Control Insulin
Fic. 6.
BSA
Tf
Control
24 hr
48 hr
72 hr
—
Lt
Fic. 5. Effects of ovotransferrin, BSA, conditioned
medium and SAF-I, on *H-thymidine incorporation
of myogenic cells. 5X10*cells/6-mm microwell
(Falcon 3072) were inoculated with 2001 of
DMEM (Dulbecco’s modified Eagle medium) con-
taining 10% horse serum. After 12 hr cultivation,
ovotransferrin (final concentration; 5 ug/ml), BSA
(final concentration; 5 ug/ml), conditioned medium
(30 wl/well) or SAF-I (5 pl/well), was added to
become final volume of 200 wl. After further 12 hr
cultivation, cells were labelled for 12 hr with 0.3 nCi
of ?H-thymidine (Amersham Co., U.K., 40-60 Ci/
mmol) in 60 “«l of DMEM, and radioactivities were
measured. Control means no addition of test sam-
ple. The data were averages of three different
cultures with SD.
Fic. 6. Effects of insulin, BSA and ovotransferrin on
3H-thymidine incorporation of the scleral fibro-
blasts. 510° cells/6-mm microwell (Falcon 3072)
were inoculated with 200 «l of DMEM containing 6
yg/ml of insulin (from bovine pancreas; No. I-1882;
Sigma Co., U.S.A.), BSA or ovotransferrin. After
cultivation for 24, 48 and 72 hr, cells were labelled
for 12 hr with 0.3 Ci of *H-thymidine and radioacti-
vities were measured. Control means no addition of
test sample. The data were averages of three
different cultures with SD.
uptake iron through membrane-bound transferrin.
In fact, melano-transferrin (p 97), a membrane-
bound transferrin like molecule, was found in
human melanoma [19]. Unfortunately, we could
not test the mechanism now, because hybridoma
(TFY004) had already attenuated.
On the other hand, Saito et al. [18] reported that
chick embryonic cells could grow without transfer-
rin in 10-100 “M Fe?*- or Fe**-ion, suggesting an
existence of unknown iron-uptake mechanism in
the chick embryonic cells. We confirmed that the
scleral fibroblasts grow even in the condition of
less than 0.2 ~M of Fe**-ion (data not shown).
So, if such iron-uptake mechanism without trans-
ferrin actually operated in the scleral fibroblasts, it
should be more enhanced.
Anatomically, the scleral fibroblasts are derived
from perichondrial tissue, and we recently found
the scleral fibroblasts possessed a capacity to dif-
ferentiate into cartilage in soft agar [20]. It is of
Cell Proliferation without Transferrin 755
interest if such nutritional characteristics as iron-
uptake without soluble transferrin are specific for
precartilagenous perichondrial cells, because they
manifest an adaptive proliferation and differentia-
tion in either skeletal remodeling during develop-
ment or fracture healing [21] under restricted
vascular environment.
ACKNOWLEDGMENTS
The authors are indebted to Dr. Makoto J. Tabata in
our laboratory for technical advices in hybridoma pro-
duction, and also to Dr. Akira Kawahara for his gener-
ous advices. The work was supported by a Grant-in-Aid
for Scientific Research to K.W. (No. 03833023; No.
03304009) from the Ministry of Education, Science and
Culture, Japan.
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ZOOLOGICAL SCIENCE 9: 757-764 (1992)
Symbiosis between Cytaeis sp. (Hydrozoa) and Niotha livescens
(Gastropoda) Starts during Their Larval Stage
Cute! Inoue! and YosHIKO KAKINUMA~
‘National Institute for Basic Biology, Laboratory of Reproduction, 38,
Nishigonaka, Myodaijicho, Okazaki, 444, and * Department of
Biology, Faculty of Science, Kagoshima University,
1-21-35, Kourimoto, Kagoshima 890, Japan
ABSTRACT—The mechanism of symbiosis between Cytaeis sp. (Hydrozoa) and Niotha livescens
(Gastropoda) has been investigated. The annual cycle of hydrozoan colonies on shells of gastropods was
observed in the field while the initiation of the symbiosis was investigated in the laboratory. In the field,
growth of colonies of polyps took place during spring, and formation of medusa buds in early summer.
From summer to autumn the buds were liberated and the polyps degenerated into stolons. Copulation
of N. livescens was observed from spring to early summer. In the laboratory, sumbiosis started at the
planula and veliger stage. The rate of adhesion and metamorphosis of planulae on newly metamor-
phosed gastropods reached a peak at 3 to 4 days after fertilization of the hydroids. The rate or reception
of one-week planulae by N. livescens was highest at the gastropodan stage just after metamorphosis.
The planulae settled on the surface of N. livescens at the base of the siphon, and then differentiated into
polyps. The metamorphosed polyps elongated their stolons over the growing juvenile gastropod, and
formed subsequent generations by asexual reproduction.
© 1992 Zoological Society of Japan
INTRODUCTION
The hydrozoan Cytaeis sp. colonizes the surface
of shells of living Niotha livescens. It has been
reported that the differentiation from polyp to
medusa of Cytaeis sp. depends on the physiological
activity of N. livescens in the breeding season [1].
However, it is still unknown when and how the
symbiosis occurs between the two species. To
investigate the mechanism of symbiosis between
the two species, we first observed their interrela-
tion in the field, as defined seasonally by the
appearance of gastropods, the formation of polyp
colonies on the shells, and the formation of medu-
sa buds from polyps. Next we investigated the
initiation of symbiosis in the laboratory.
Accepted June 2, 1992
Received April 8, 1992
" To whom reprint requests should be addressed.
MATERIALS AND METHODS
Field observations
Gastropods, Niotha livescens, covered with a
colony of the hydroids, Cytaeis sp. (Fig. 1B), were
collected from the sand shore of Yojiro-ga-hama in
Kagoshima city (Kagoshima Pref., Japan). Gas-
tropods were captured with fresh fish pieces at
several points on the shore every month (Fig. 1A).
Within 30 minutes about 20 to 50 gastropods
gathered around the bait. Of these, the number of
gastropods with a colony of hydrozoa averaged 5
to 7. The pattern of colony distribution and the
number of polyps and/or medusa buds were re-
corded on schematic drawings of the shell and are
summarized in Fig. 2. The animals were released
at the same points of capture during following day.
Laboratory observations
Gastropods with a colony of hydrozoa were
cultured in laboraotry aquaria with circulating
seawater of 25°C. N. livescens were fed with fish
and Cytaeis sp. with brine shrimp. Freshly
Fic.
Number of polyps and medusa buds
Fic.
1. The hydrozoan colony on shells of N. livescens.
A. The gastropods were trapped with fish meat.
Scale, 1 cm, B. Hydrozoan colony on a shell. Scale,
1 mm.
nN
oO
Temperature (°C)
15
400 tl 10
300
200
100
0
JFMAMJJASOND J FM
1985 1986
2. Annual reproductive cycle of Cylaeis sp. and N.
livescens. The largest number of polyps or medusa
buds on captured gastropoods of each collection was
plotted. Ordinate left; Number of polyps (—@—),
medusa buds (—O—), and right, temperature
(—A—). Abscissa: Months in which gastropods
were collected. N. /. (@), breeding season of N.
livescens.
C. INOUE AND Y. KAKINUMA
spawned eggs from N. livescens (3 individuals)
were collected from the walls of the auqgaria (about
20 liters) and transferred to petri dishes where they
were allowed to develop into veligers and were
then hatched.
As soon as the gastropods spawned eggs,
another group of adult gastropods carrying col-
onies of polyps were transferred to petri dishes
(about 8cm in height) and kept at 25°C. They
were fed brine shrimps to obtain hydrozoan medu-
sae. Newly released medusae were also fed on
brine shrimp. Spawning was induced by exposing
mature medusa, previously kept in the dark, to
light. Fertilization took place without further
treatment. The resulting fertilized eggs were
allowed to develop, and subsequent planula larvae
were maintained and used in the experiments.
The behavior of planula larvae, young polyps,
veligers and juvenile gastropods after the estab-
lishment of symbiosis was continuously observed.
In order to examine the ability of attachment of
planulae, one gastropod was mixed with 6 planulae
of the same age, just after metamorphosis and the
number of attached planulae was counted within
10 days (Fig. 4). Six trials were carried out, each
with planulae of a different age. Seven-eight hr old
planulae were designated as 0-day planula. In
order to examine the rate of reception of 1-week
planula by N. livescens of various ages, six 1-week
planulae were mixed with one gastropod of vari-
able age (Fig. 5, arrows: time point of mixing).
The number planulae found to be attached within
10 days was counted. Six trials, each with gastro-
pods of a different age were carried out.
Cytological examinations of planulae
Planulae were fixed in glutaraldehyde (2% in sea
water) for 1 hour at room temperature. They were
dehydrated in graded ethanol series, embedded in
JB-4 plastic medium (Polyscience Inc.), and sec-
tioned at 1-2 um with glass knives. The sections
were stained routinely with Delafield’s hematoxy-
lin and eosin and observed under a light micro-
scope.
Symbiosis between Hydrozoa and Gastropoda 169)
RESULTS
Coexistence of the two species in the field
The number of polyps varied between gastro-
pods, and fluctuated throughout the year. The
maximum number of polyps and medusa buds for
each observation through the year are shown in
Fig. 2. In January of 1985 no polyp was observed.
In February and April, a total of 47 and 90 polyps
were recorded respectively. In May, the polyps
(336) covered most of the surface of the shells, and
122 medusa buds were counted. In June and early
July, the number of polyps was 255 and 234,
respectively. During the same period, 160 and 132
medusa buds were counted. In late July, the
number of polyps had decreased to 4. Medusa
buds were not observed. These results show that
the colony of polyps degenerates from autumn to
winter, and appear again in early spring. From
spring tc summer, they grow and cover the surface
of shells. Medusa buds are formed during early
summer and are probably released in the same
period. From summer to winter, the number of
polyps fluctuate, but medusa buds are not
observed in this period. WN. livescens displayed
copulation from spring to early summer, which
coincided with the appearance of hydrozoan
medusae.
Interrelation of the two species in the laboratory
Egg-laying and the development of N. livescens
The total number of spawned eggs collected was
722 from May to July, 1985, and about 300 from
July to August, 1986. The diameter of the egg
capsule was about 350 ~m. Embryos inside the egg
capsules developed normally (larval shells, vela,
eyes, feet, and siphons).
Embryos hatched and developed into veliger
larvae about 15 days after egg-laying (25°C). The
earliest veliger, with a small velum and no siphon,
was observed on the 9th day after egg-laying.
Veligers in this stage displayed only planktonic
(swimming) behavior and were designated as early
veligers. Veligers 15 to 16 day old were observed
to have a broad velum and a siphon, and were
designated as late veliger. From 17 to 30 days after
egg-laying, they metamorphosed to juvenile gas-
tropods, revealed by withered vela, and entered
into completely benthonic life.
Development of Cytaeis sp.
Colonies of Cytaeis sp. formed medusa buds
during the breeding season of N. livescens. The
medusa buds grew for about 1 week up to 1 mm in
diameter and then separated from the colony.
About 4 days after their release (25°C) they
spawned gametes in early morning. The diameter
of the eggs was about 100 um.
The living planulae (Fig. 3A) and the sections
(Fig. 3B) gave the following observations. At 7 to
8 hr after spawning (20°C), the planulae were just
after hatching. Blastocoels were still present and
differentiation of endoderm by polarization at the
posterior ectoderm had begun to occur. The total
number of the cells was small. In 1-day planulae,
blastocoels had disappeared, and differentiation of
endoderm from ectoderm was completed. How-
ever, nematocysts were not observed. The planu-
lae began to swim at this stage. In 4-day planulae,
the nematocysts were recognized at the vegetal
pole. The planulae swam most actively in this
stage, whilst the number of cells was highest. The
cells contained many secretions which stained red
with eosin. Twenty days after spawning, the size of
planulae and the number of cells decreased and
vacuoles were observed in the endoderm. Sixty
days after spawning, the planulae gradually be-
came motionless and were observed to be lying on
the surface of the sediment. The shape of
ectodermal cells became flat and the endoderm
degraded with a large central vacuole.
Rate of attachment of planulae of various ages on
newly metamorphosed N. livescens
Planulae did not attach to shells within the first 1
or 2 days after mixing (Fig. 4). The rate of
attachment of 0-day planula was 40%. The highest
rate of attachment was obtained with 4-day planu-
lae. At this age almost all planulae were attached
to the shells within a few days after mixing.
Ten-day planulae still showed a high rate of attach-
ment, but the rate decreased rapidly with increas-
ing age of planula. Sixty-day planulae did not
show any capability of attaching to shells.
760 C. INOUE AND Y. KAKINUMA
Fic. 3. Early development of Cyraeis sp. A. Living planulae (bright-field micrographs). B. l-m sections of
planulae (hematoxylin-eosin stained). 1, 2, 3, 4, and 5. 7-8 hours, 1 day, 4 days, 20 days, and 60 days after
fertilization, respectively. Scale, 50 «m.
Symbiosis between Hydrozoa and Gastropoda
—_
00
50
Attachment of planulae (%)
0 10 20 30 40 50 60
Age of planulae (days)
Fic. 4. Rate of attachment of planulae of different ages
on newly metamorphosed gastropods. Ordinate:
Attachment of planulae (%). Abscissa: The age of
planulae (days). One gastropod just after meta-
morphosis was mixed with six planulae at different
ages.
Rate of reception of I-week planula by N. livescens
shell of various ages
The veliger stage of N. livescens was observed to
begin on the ninth day after egg-laying (Fig. 5, A)
while metamorphosis took place about 1 week
after the beginning of the veliger stage (Fig. 5, B).
The oldest veliger was observed 27 days after
egg-laying (Fig.5, C). Therefore, the period of
metamorphosis could be regarded as the double-
shadowed area shown in Fig. 5 (between B and C).
When the planulae were mixed with 10-day gastro-
A B Cc
~
109
“Lt hy
Reception of planulae (%)
ao
(=)
i Yj
Age of gastropods (days)
Fic.5. Rate of reception of one-week planulae by
gastropods of various ages. Six l-week planulae
were mixed with one gastropod of various ages.
Ordinate: Reception of planulae (%). Abscissa:
The age of the gastropods (days). Arrows indicate
time point of mixing.
761
pods, the rate of reception was 40%. In this case,
no planulae attached to the shell within the first 5
days after mixing. Consequently, the data-plotting
should not have begun until the 15th day after egg
laying, and the data were therefore re-plotted with
open cricles. The highest rate or reception was
observed during the period of metamorphosis.
The rate of reception remained at 50% throughout
the early juvenile stages.
Settlement, metamorphosis and colony formation
of Cytaeis sp.
As soon as the juvenile gastropods and planulae
encountered each other, the gastropods showed an
active movement and then immediately became
motionless. On the other hand, the planulae
displayed position-searching behavior, initially
creeping around the siphon and finally selectively
settling at the base of the siphon (Fig. 6). Most
planulae settled on this place of the shell.
1
Fic. 6. The location of settlement of planulae on young
gastropods. Note the display of position-seaching
behavior by planula on a motionless gastropod.
Scale, 100 un.
The planulae metamorphosed into a polyp with-
in 4 days after settlement on the shell (Fig. 7). The
process of the metamorphosis was divided into 5
C. INOUE AND Y. KakINUMA
Rate of metamorphosed hydrozoa (%)
(3)O0 @o 00 oe 9
, ’ ’ . ff ate 1 '¢ a
Ge TR 4 we tae Ar
‘ tf uw
4 ,
’
ve
s
.
‘
‘
S
5
2 3
1
Days after attachment
Rate of metamorphosed individuals was expressed as %.
Fic. 7. Metamorphosis of Cytaeis sp. on the shell.
Ordinate: Stages of metamorphosis of hydrozoa. Abscissa: Days after attachment. Scale, 100 pm. I-V. Stages of
metamorphosis (see text).
26 28 (days)
18
Formation of the polyp colony on the shell. Numbers indicate days after attachment of planula on the shell.
1 8
Fic. 8.
Scale, 1 mm.
Symbiosis between Hydrozoa and Gastropoda 763
stages: 1; up to just after settlement, 2; beginning
of formation of the gastric cavity, 3; completion of
the gastric cavity, 4; formation of tentacle rudi-
ments, and 5; completion of metamorphosis to a
polyp with tentacles. Typically (43% of observa-
tions), a planulae metamorphosed to a polyp with-
in 2 days along the course shown by the thickened
line in Fig. 7. When planulae were cultured in a
petri dish, almost all of the planulae were on the
bottom of the dish and died. Only a few planulae
differentiated into a polyp (about 0.01%).
As mentioned above a polyp initially located at
the base of the siphon. This polyp elongated a
stolon (initial one) in the direction of the shell
growth. The second polyp was then formed, again
at the base of the siphon, around 15 days after the
first planula settlement. After the second polyp
formation, the initial polyp degenerated and be-
came engulfed by the growth of the shell (Fig. 8).
Thus, the metamorphosed polyp produced subse-
qunet generations of the polyp colony asexually.
Food was considered to be an important factor for
the asexual reproduction of Cytaeis sp., because
lack of food (brine shrimp, in the present study)
hindered polyp formation.
Rate of survival and growth of N. livescens
The symbiotic relationship and the rate of sur-
vival of the gastropods were examined ex-
perimentally. First, veligers were mixed with
1-week planulae. Out of 86 veligers, the number
of veligers on which planula settled was 18, of
which 14 veligers metamorphosed and grew while
4 died before metamorphosis. Of the remaining 68
veligers, on which planulae did not settle, 18
veligers died before metamorphosis while 50 veli-
gers metamorphosed and grew.
After metamorphosis, a second trial was carried
out, in which gastropods which were still without
hydrozoa were mixed with 1-week planulae.
Among 50 gastropods, the number of gastropods
which were settled with planula was 37 and these
gastropods all survived. Among the rest, i.e. 13
gastropods on which planulae did not settle, 9
gastropods died within 10 days and gastropods
grew (Fig. 9). The survival rate of the gastropods
with VP* (Veligers with planulae) and GP*
(young gastropods with planulae) or without VP~
37)
GP +,100%)
|
4(GP ,30%)
Number of veligers or gastropods
o
o
Lo
|
<+——Veliger stage ————> | ~~— Young gastropod stage —>
Metamorphosis
Fic. 9. Symbiotic relationship and the rate of survival
of the gastropods. VP*: Veligers with planulae,
VP: Veligers without planulae, GP*: young gas-
tropods with planulae, GP: young gastropods with-
out planulae. Ordinate: length of each line (i.e.
numeral on each line) indicates numbers of veligers
or gastropods. Abscissa: stage of the gastropod.
a
(Veligers without planulae) and GP (young gas-
tropods without planulae) was estimated as 89%
[(77% + 100%) /2] and 52% [(73% +30%)/2], re-
spectively. If the calculation is simply done at the
last observation (i.e. the right-most line of Fig. 9),
the rate of survival of gastropods without hydroids
is about 7% [4/(14+37+4) x 100].
DISCUSSION
In the laboratory symbiosis took place between
planula and late veliger stage respectively. The
ability of planulae to go through metamorphosis
appears to be maintained for about 20 days and
then gradually disappears. This in turn implies
that suppression of metamorphosis also lasts for
this period provided a stimulation for triggering
metamorphosis is not given.
As for stimulation, it has been reported that in
Hydractinia echinata the stimulus causing initiation
of metamorphosis emanates from certain marine
bacteria [2]. The stimulus for Cytaeis sp. may also
be a chemical factor, because the planulae of
Cytaeis sp. always settle at the base of the siphon
of N. livescens.
It is considered that a receptive system for a
metamorpnosis-triggering factor must exist in
planulae. It has been reported that neural dif-
764 C. INOUE AND Y. KAKINUMA
ferentiation begins in the planular ectoderm 24
hours after fertilization and continues throughout
larval development [3, 4]. One reason explaining
the inability of 60-day planulae to attach is consi-
dered to be a lack of swimming ability which
reduces the chance for planulae to encounter the
gastropods. It is also possible that in 60 day
planulae a degeneration of the neural system re-
duces their ability to perceive a stimulation factor.
As mentioned before, a few planulae differenti-
ated into a polyp when cultured in a petri dish. It is
considered that metamorphosis of Cytaeis. sp. can
be triggered by other factors, and once triggered,
metamorphosis seems to progress without any
further exogenous stimulation. However, in the
field we have never seen a colony of Cytaeis sp.
except on the surface of the N. livescens.
The larvae of N. livescens clearly offer a substra-
tum on which the planulae adhere and meta-
morphose. Since the planulae and the juvenile
gastropod display specific behavior as soon as a
planulae and a juvenile gastropod encounter each
other, and since the position of planula settlement
is always at the base of the siphon, we can specu-
late that the gastropods secrete some planula-
attacting substance from the base of the siphon, to
which the palnula reacts. On the other hand, the
hydrozoan planula may secrete some factor to
make the gastropod motionless.
Although one trial was not enough to determine
the survival rate, the rate of survival of gastropods
that are attached by planula tends to be higher
than that of gastropods without planulae. There-
fore the adhesion of planula may provide some
adequate condition for the gastropodan physiology
as Hirai and Kakinuma previously reported for the
adult sage [1, 5]. Among the collected gastropods,
the percentage of gastropods without hydrozoa is
fairly high (about 70%). And much higher than
calculated from the laboratory trials (finally about
7%). Therefore, in the laboratory, symbiosis may
be preferable for the gastropod particularly in the
juvenile stage.
Since in the field the breeding seasons of the two
species coincide with each other, symbiosis be-
tween the two species must take place at the larval
stage, although the gastropods can survive without
hydrozoa. Whether the loose symbiosis between
these species inclines to a more strict relationship
or declines towards dissolution requires long term
observation. For the hydroids, however, the cur-
rent relation seems to be necessary to survive and
to diverse in the field.
ACKNOWLEDGMENTS
We thanks Dr. H. Shirai (Okayama University) for
critical reading of the manuscript, Messrs. M. Tabata and
Y. Ikimori (Kagoshima Marine Park) for help in collect-
ing the gastropods, and Dr. J. Komen and Dr. Cocket
(National Institute for Basic Biology) for reading the
manuscript.
REFERENCES
1 Hirai, E. and Kakinuma, Y. (1971) On symbiotic
relations between differentiation of a hydrozoan
Cytaeis uchidae with a gastropod Niotha livescens.
Bull. Mar. Biol. Stat. Asamushi, 14: 65-77.
2 Spindler, K. D. and Muller, W. A. (1972) Induction
of metamorphosis by bacteria and by a lithium-pulse
in the larvae of Hydractinia echinata (Hydrozoa).
Wilhelm Roux’ Archiv 169: 271-280.
3 Martin, V. J. (1988) Development of nerve cells in
hydrozoan planulae: I. Differentiation of ganglionic
cells. Biol. Bull. 174: 319-329.
4 Martin, V. J. (1988) Development of nerve cells in
hydrozoan planulae: II. Examination of sensory cell
differentiation using electron microscopy and im-
munocytochemistry. Biol. Bull. 175: 65-78.
5 Hirai, E. and Kakinuma, Y. (1973) Differentiation
and symbiosis in two hydrozoans. Publications of the
Seto Marine Biological Laboratory, 20: 257-273.
ZOOLOGICAL SCIENCE 9: 765-773 (1992)
Effects of Olfactory Tract Section on Brain GnRH Distribution,
Plasma Gonadotropin Levels, and Gonadal Stage in Goldfish
Maxito Kopayasui!, MAsSAFUMI AMANO!, YoOSHIHISA HASEGAWA2
Koicut Okuzawa? and Katsumi Arpa!
‘Department of Fisheries, Faculty of Agriculture, University of Tokyo,
Bunkyo, Tokyo 113, *Department of Obstetrics and Gynecology,
Gunma University, School of Medicine, Maebashi, Gunma 321-16,
and *Inland Station, National Research Institute of Aquaculture,
Tamaki, Watarai, Mie 519-04, Japan
ABSTRACT— Goldfish in early sexual recrudescence were olfactory tract-sectioned (OTX) or sham-
operated in order to examine the distribution of brain gonadotropin releasing-hormone (GnRH)
originating from the terminal nerve (IN), and the involvement of GnRH in reproduction. Fish were
kept at 20°C, and one month following surgery, brain contents of two types of GnRHs, salmon type
(sGnRH) and chicken-II type (CGnRH-II), were measured by specific radioimmunoassays. Plasma
gonadotropin (GTH) levels and the stages of gonadal development were also determined. After OTX,
sGnRH contents showed a marked decrease in all brain areas (telencephalon, hypothalamus, optic
tectum-thalamus, cerebellum, medulla oblongata) except in the olfactory bulbs, whereas cGnRH-II
contents in the brain and the pituitary gland showed no clear changes. Pituitary sGnRH contents and
plasma GTH levels showed slight decreases in OTX males, but not in females. In spite of large
decreases in brain sGnRH contents, gonadal development in OTX fish was as active as that in
sham-operated fish with the progression of spermatogenesis in males and vitellogenesis in females.
These results suggest that most of the sGnRH in the brain originates from the TN, and that TN-GnRH is
© 1992 Zoological Society of Japan
not essential to gonadal development in goldfish.
INTRODUCTION
Gonadotropin releasing-hormone (GnRH) is a
decapeptide which regulates reproductive activi-
ties, such as the stimulation of gonadotropin
(GTH) release from the pituitary gland [1] and the
potentiation of sexual behavior [2]. Recent studies
have shown that more than one type of GnRH
exists in the brain of teleosts, and most of the
species examined possess salmon type (sGnRH)
and chicken-II type (CGnRH-II) [3]. Immunocyto-
chemical studies revealed that major populations
of GnRH-producing cells in teleosts are localized
in the preoptic area, the terminal nerve (TN,
homologous to the nucleus olfactoretinalis), the
midbrain tegmentum, and the nucleus lateralis
tubelis [4-12], although particular GnRH type was
Accepted April 19, 1992
Received February 14, 1992
not necessarily examined in all the cases, in con-
text of the antibodies used in each study.
Among these GnRH populations, the GnRH
system in the preoptic area is considered to be
morphologically equivalent to that of the pre-
optico-infundibular pathway of other vertebrates
[13]. The GnRH in this system is presumed to
function as a hypophysiotropic hormone in the
stimulation of GTH release [7]. However, direct
evidence of the release of GnRH from this system
has not been obtained in teleosts. Since teleosts
lack the hypothalamo-pituitary portal system, the
measurement of GnRH near its site of release is
quite difficult.
Many studies on the TN system in teleosts have
been conducted regarding morphological aspects
[14], but the physiological role of the system is not
fully understood. Demski and Northcutt [15]
suggested that the TN system mediates the be-
havioral response to sex pheromones in goldfish.
766 M. KosayAsut, M. AMANo et al.
However, this possibility now seems less probable,
since it has been demonstrated that chemosensory
responses to sex pheromones in the goldfish are
mediated by the medial olfactory system instead of
the TN system [16].
Although the TN system is known to produce
GnRH, it is not clear whether GnRH originating
from the TN is involved in gonadal maturation
through GTH secretion. There are some studies
on changes in brain GnRH contents in relation to
gonadal maturation. However, the results of these
studies are not consistent. In caribe colorado,
Pygocentrus notatus [17] and masu_ salmon,
Oncorhynchus masou [18], brain GnRH contents
(sGnRH in masu salmon) were high when gonads
were mature, but no clear correlation was
observed between brain GnRH contents and
gonadal maturity in eel, Anguilla anguilla [19],
goldfish, Carassius auratus [20] and rainbow trout,
Oncorhynchus mykiss [21]. In brown trout, Salmo
trutta [22], a correlation was observed between
pituitary GnRH contents and plasma GTH levels.
These studies measured brain and pituitary GnRH
contents without considering the origin of GnRH
fibers, and moreover, most of these studies were
conducted using radioimmunoassay (RIA) whose
antibodies were not specific to different types of
teleost GnRH.
In the goldfish, two types of GnRH, sGnRH and
cGnRH-II, were chromatographically and im-
munologically identified in the brain [23]. Major
populations of GnRH-producing cells were found
in the IN, preoptic area, midbrain tegmentum,
and nucleus lateralis tubelis by immunocyto-
chemical studies [4, 7]. Since the olfactory bulbs of
goldfish are of the pedunculated type with elon-
gated olfactory tracts, we can easily block the
axonal transport of GnRH from the TN to other
brain regions by sectioning the olfactory tracts.
In the present study, the effects of olfactory tract
section were studied in goldfish in order to ex-
amine the brain distribution of GnRH of the TN.
Brain GnRH was measured by RIAs using highly
specific antibodies to sGnRH and cGnRH-II [21].
Plasma gonadotropin (GTH) and gonadal de-
velopmental stages were also determined in order
to examine whether TN-GnRH was involved in
gonadal development.
MATERIALS AND METHODS
Goldfish
Goldfish were obtained from a commercial
source and kept in stock tanks under natural
condition. Fish weighing 30-40 g were used for
the experiment.
Experimental Protocol
Fish in early sexual recrudcence were olfactory
tract-sectioned (male N=13; female, N=7) or
sham-operated (male, N=13; female, N=5) on
October 19. Initial body weights of each group
were as follows (mean+SEM g): male; sham 34.9
+0.8, OTX 33.7+0.8: female; sham 32.1+1.8,
OTX 32.4+1.9. The experiment was originally
designed to use males since the involvement of
olfactory system in reproduction has been reported
mostly in male goldfish [15, 24, 25]. Female groups
were supplemtned as an additional part of the
experiment, although sample size was small.
For olfactory tract section, four-sided flap was
cut in the frontal bone using a disc saw. The first
cut was made aligned with the center of the iris. If
this cut were made more posteriorly, damage to
the pineal gland would be likely to occur. The
second cut, parallel to the first, was made just
behind the nares. The third and fourth cuts,
parallel to the longitudinal axis of the fish, con-
nected to the first and the second cuts. After
removal of a square flap of the frontal bone, fat
and cranial fluid were gently wiped out with paper.
The olfactory tracts were bilaterally sectioned at
two places with Wecker’s scissors (Fig. 1), and the
resultant sections were removed to prevent regen-
eration of the olfactory tract fibers (OTX fish).
The cavity resulting from the operation was filled
with gelatin sponge (Spongel, Yamanouchi Phar-
maceutical Co.). A sham operation was per-
formed in the same manner without cutting the
olfactory tracts (sham-operated fish).
After the operation, fish were kept in 25 ppm
oxyteracyclin (Sigma) overnight, and then trans-
ferred to a 60 liter experimental tank (males and
females in the same tank) which was kept at 20°C
under ISL (lights on at 0500 hr). Fish were fed
with commercial trout pellets once a day. Fish
Brain GnRH in OTX Goldfish 767
were sampled on November 20. After being
anesthetized with 0.02% tricaine methansulfonate,
their body weights were measured. Blood samples
were taken from caudal vasculature with a hepari-
nized needle and syringe. Fish were sacrificed by
decapitation, and the brain and pituitary glands
were collected. The brains were dissected into six
parts (olfactory bulbs, telencephalon, hypothala-
mus, optic tectum-thalamus, cerebellum, medulla
oblongata) as shown in Fig. 1. The tissues were
frozen on dry ice after being weighed. The gonads
were dissected out and weighed to determine the
gonadosomatic index (GSI). For histological
observation, the gonads were fixed with Bouin’s
solution and embedded in paraffin. Sections were
stained with hematoxylin and eosin. Ovarian
developmental stages were identified according to
the classification by Yamamoto and Yamazaki
[26].
PIT
Fic. 1. Schematic diagram of a sagittal section of
goldfish brain showing sites of severance of the
olfactory tracts (arrows) and disection of the brain
for the determination of GnRH. OLT, olfactory
tracts; OB, olfactory bulbs; TEL, telencephalon,
including preoptic area; OT-THAL, optic tectum-
thalamus, including midbrain tegmentum; CER,
cerebellum; MED, medulla oblongata; PIT,
pituitary.
RIA
Salmon GnRH and cGnRH-II were measured
by specific RIAs as described by Okuzawa et al.
[21]. The cross-reactivity of cGnRH-II in the
sGnRH RIA (antibody to sGnRH, Lot No. 2) was
1.58%, and the cross-reactivity of sGnRH in the
cGnRH-II RIA (antibody to cGnRH-II, Lot R-E)
was 0.051%.
Measurement of GnRH in the goldfish brain by
the RIA was validated by examing parallelism of
the displacement curves of goldfish brain extract.
High performance liquid chromatography (HPLC)
analysis followed by RIA was also employed for
the validation of the RIA. To test parallelism,
goldfish brain tissue was homogenized by sonica-
tion in 0.1 N HCl. The homogenate was centri-
fuged at 10,000 xg for 30 min at 4°C. The super-
natant was frozen, lyophilized, and reconstituted
in assay buffer. The supernatant obtained after
centrifuging again at 10,000 g for 30 min at 4°C
was used for the GnRH RIAs. The brain extract
of goldfish produced displacement curves which
Salmon GnRH RIA
100
Dilution of brain extract
_———_—— el
Brain extract
= 50 Salmon GnRH
{e)
gl
a
0
9.8 39 156 625 (pg/ml)
Chicken GnRH-Il RIA
100 Dilution of brain extract
Brain extract
SS
= 501 Chicken GnRH-II
a
oO
9.8 39 156 625
(pg/ml)
Fic. 2. Top. Competition curves for salmon GnRH and
brain extract of goldfish in the salmon GnRH RIA.
Bottom. Competition curves for chicken GnRH-II
and brain extract of goldfish in the chicken GnRH-II
RIA. The scale for dilution of brain extract indi-
cates a twofold serial dilution. Each point repre-
sents the mean of duplicate determinations.
768 M. Kopayasui, M. AMANO et al.
were parallel to sGnRH and cGnRH-II standards
in respective RIAs (Fig. 2). For the measurement
of brain GnRH contents of the experimental fish,
the brains were extracted in the same method as
for the paralellism test.
For HPLC analysis, the brain was extracted as
described by Okuzawa et al. [21]. The extract was
injected through a 2 ml-injection loop onto a TSK-
ODS 80TM column (0.4625 cm, Tosoh Co.
Ltd., Tokyo, Japan). A Hitachi high performance
liquid chromatography (Model 655 A) was pro-
grammed for a flow rate of 1 ml/min. The extract
was applied at the beginning of a 10-min isocratic
period of 17% acetonitrile (CH3CN) in 0.1%
trifluoroacetic acid; CH3CN was then increased to
27% over a 10-min period. GnRHs were eluted
under isocratic conditions at 27% CN3CN. Frac-
tions (1 ml) were collected and aliquots were
assayed for GnRH. HPLC analysis revealed that
goldfish brain contained a peptide chromato-
graphically and immunologically identical to
sGnRH and a peptide identical to cGnRH-II (Fig.
3). Although there was some immunoreactive
materials which were chromatographycally diffe-
rent from sGnRH and cGNRH-II in the goldfish
brain, their amount was considered negligible, and
therefore it was not necessary to separate sGnRH
and cGnRH-II by HPLC prior to the RIAs.
Plasma GTH levels were measured using silver
carp GTH RIA as described by Kobayashi et al.
[27]. Purified silver carp GTH (Lot SS) was used
as a standard and for iodination [28], and an
antibody (Lot No. 1) raised against the silver carp
GTH (Lot SS) was used for the RIA [29]. Valida-
tion of the RIA system for use in goldfish was
achieved by obtaining parallelism for a serial dilu-
tion of plasma samples collected from ovulated
females. Recoveries at three different doses, high,
middle, and low, were 95.2, 102.2, and 115.4%,
respectively. The GIH measured in this study is
considered to be equivalent to GTH II according
to Kawauchi’s designation [30] by physicochemical
characters of the silver carp GTH [28], an im-
munocytochemical study of goldfish pituitary GTH
cells using the silver carp GTH antibody (Lot No.
3) [29], and plasma profiles of GTH during ovula-
tion in goldfish measured by the silver carp GTH
RIA [31].
Salmon GnRH RIA
Salmon
Chicken-ll
4
>
8
5 3
3 3
= S
z= Chicken GnRH-II RIA H
5 I
I
Elution time (min)
Fic. 3. Reverse-phase HPLC of goldfish brain extract
followed by salmon GnRH RIA (top) and chicken
GnRH-II RIA (bottom). Arrows indicate the elu-
tion time of synthetic chicken GnRH-II and salmon
GnRH. The mobile phase was CH3CN containing
0.1% TFA.
Statistics
Statistical analysis of the results were done by
Student’s t-test or Cochran-Cox method.
RESULTS
One month following surgery, the experimental
fish exhibited gains in body weight; and there were
no significant differences in final body weights
between sham-operated and OTX fish (mean+
SEM g): male; sham 42.6+0.7, OTX 43.7+1.4;
female; sham 41.8+2.8, OTX 44.4+2.2 (Fig. 4).
After OTX, sGnRH contents in OTX fish of
both sexes showed a marked decrease (P<0.01) in
all brain areas (telencephalon, hypothalamus,
optictectum-thalamus, cerebellum, medulla oblon-
gata) except in the olfactory bulbs (Fig. 5 and 6).
Chicken GnRH-II contents in OTX fish showed a
tendency toward decrease, but only the decrease in
hypothalamus of males was significant (P<0.05).
Brain GnRH in OTX Goldfish 769
Male Female
Body weight
60 y g
40
i)
20
S
A S
N 13 13 7
GSI
4
3
& 2
if
S
A S
N 12 13 5 7
Plasma GTH
20
= *
ro.)
Cc
— 10
S S
0
N 12 12 BY
Fic. 4. Effects of olfactory tract section on body
weight, gonadosomatic index (GSI), and plasma
levels of gonadotropin (GTH) in goldfish. S, sham-
operated fish; O, olfactory tract-sectioned fish.
Each column represents the mean and SEM. Level
of significance; *, P<0.05.
Salmon GnRH contents in the olfactory bulbs
showed no significant difference between sham-
operated and OTX fish in both sexes. Chicken
GnRH-II contents of the olfactory bulbs were
mostly low and no clear change was detected after
the operation.
Pituitary sGnRH contents showed decreased
levels in OTX males (P<0.05), but not in females.
Pituitary cGnRH-II contents showed no significant
changes after the operation in both males and
females.
Plasma GTH levels in OTX males were slightly
lower (P<0.05) than those in sham-operated
males, but no significant difference was observed
in females (Fig. 4).
There was no significant differences in GSI
values between OTX and sham-operated fish in
both sexes (Fig.4). Histological observation
showed that under conditions which are favorable
for gonadal maturation in goldfish (20°C, 15L)
both OTX and sham-operated fish had developing
gonads regardless of the difference in brain GnRH
contents. All males had spermatogenic testes and
lobules filled with spermatozoa. The oocytes of
the ovaries in all females had advanced to the
primary yolk stage except one OTX fish which had
the ovaries with secondary yolk stage oocytes.
DISCUSSION
The brain GnRH distribution and contents in
sham-operated goldfish showed a pattern similar to
those of goldfish reported in other studies [20, 23]
and those of salmonid fishes [18, 21]: Salmon
GnRH was distributed in a larger amount in the
olfactory bulbs, telencephalon, hypothalamus, and
the pituitary gland than in other regions, whereas
cGnRH-II was distributed widely throughout the
brain with the highest concentration in the medulla
oblongata. The difference between goldfish and
salmonid fishes is that the goldfish pituitary con-
tains cGnRH-II, which is absent in salmonid
pituitaries [12, 18, 21].
After OTX, brain sGnRH contents showed a
marked derease in all brain areas except in the
olfactory bulbs. These results suggest that sGnRH
in the goldfish brain mostly originates from the
TN. That is, sGnRH contents in various brain
regions reflect sGnRH in fibers which are trans-
ported from the TN cell bodies in the olfactory
bulbs. Immunocytochemical studies in other spe-
cies support this hypothesis. In the catfish, Clarias
batrachus [9] and the dwarf gourami, Colisa lalia
[11] GnRH fibers from the TN send projections to
various regions of brain parts. Electrical lesion of
the TN cells in gourami caused disappearance of
most of immunoreactive fibers in the brain leaving
GnRH cells and fibers in preoptic area intact [32].
Furthermore, it has been also demonstrated by
770
GnRH (ng/mg tissue)
M. KopayasHi, M. AMANO et al.
OB TEL HYP OT CER MED PIT
THAL
Salmon GnRH
70
60 150
50
40 100
30 as
20 50
10
0
s O
Chicken GnRH-II
Fic.5. Effects of olfactory tract section on salmon GnRH and chicken GnRH-II concentrations in discrete brain
areas in male goldfish. OB, olfactory bulbs; TEL, telencephalon, including preoptic area; OT-THAL, optic
tectum-thalamus, including midbrain tegmentum; CER, cerebellum; MED, medulla oblongata; PIT, pituitary.
S, sham-operated fish (N=12); O, olfactory tract-sectioned fish (N=13). ND, Nondetectable. Each column
represents the mean and SEM. Level of significance: *, P<0.05; **, P<0.01; ***, P<0.001.
GnRH (ng/mg tissue)
OB TEL HYP OT CER MED PIT
THAL
Salmon GnRH
40 100
30 15
20 10 50
10 5 * * * *
* * * *
0 0 0
Ss oO Ss Oo Ss Oo s O Ss Oo s Oo
Chicken GnRH-II
Fic. 6. Effects of olfactory tract section on salmon GnRH and chicken GnRH-II concentrations in discrete brain
areas in female goldfish. Abbreviations and symbols as in Fig. 5. S, N=5; O, N=7.
Brain GnRH in OTX Goldfish 771
intracellular staining in gourami that a single TN-
GnRH cell projects to all brain regions where
GnRH fibers has been demonstrated but not to the
pituitary [33]. Other immunocytochemical studies
are also suggestive of the existence of projections
of GnRH fibers from the TN to other brain regions
[5, 7]. It seems likely that a common anatomical
feature in teleosts is projection by the TN system
of some type of GnRH to various regions of the
brain, although the physiological function of such
GnRH remains to be elucidated.
It is not clear from the present study whether TN
cells of goldfish produce cGnRH-II. Chicken
GnRH-II contents were mostly undetectable in the
olfactory bulbs and showed no clear changes after
OTX in the other brain areas except in the
hypothalamus of males. In masu salmon, cell
bodies of cGnRH-II neurons were localized in the
midbrain tegmentum, and only a few fibers were
found in the olfactory bulbs [12]. Small amounts of
cGnRH-II in the goldfish olfactorty bulbs mea-
sured by RIA may be provided by fibers from
other regions of the brain, such as the midbrain
tegmentum. To confirm the anatomical distribu-
tion of cell bodies and fibers of sGnRH and
cGnRH-II immunocytochemical
approach is required.
Although OTX caused a marked decrease in
brain sGnRH contents, no gonadal regession was
observed both in males and females in contrast to
the example of hypophysectomized female goldfish
[34]. There was no difference in the stages of
gonadal development between OTX and sham-
operated fish. Under conditions which enhance
gonadal maturation of goldfish, fish of both groups
showed active gonadal development which would
not normally occur during non-spawning period.
These results suggest that TN-sGnRH is not essen-
tial to gonadal development in goldfish. Although
abnormal changes were seen in the gonads after
lesion of nucleus olfactoretinalis in platyfish,
Xiphophorus maculatus [8], reasons for different
responses to a similar treatment between the two
species is unclear.
OTX males showed decreased levels of pituitary
sGnRH which may be responsible for the decrease
in plasma GTH levels. There are some possible
interpretations for this decrease, such as blockade
neurons, an
of a direct innervation of sGnRH fibers from the
TN to the pituitary gland, or a removal of stimula-
tion to sGnRH neurons which send fibers to the
pituitary, etc. 17a,20-Dihydroxy-4-pregnen-3-
one, one of the goldfish sex pheromones, is known
to stimulate GTH secretion in males during spawn-
ing [35]. If female goldfish release sex pheromones
which stimulate basal GTH release in males, the
decrease in the pituitary sGnRH contents and
plasma GTH levels in OTX males may be partly
explained by inability in receiving sex pheromones
since OTX fish were anosmic in the present study.
However, a decrease in pituitary sGnRH contents
and plasma GTH levels to the extent observed in
this study did not seem to affect basal testicular
development.
As well as the gonadal stages, no differences
were observed in body weight between OTX and
sham-operated fish. Reduction of the number of
feeding acts (bites directed toward gravel and
artificial vegetation) after OTX is reported in
goldfish [24], but the actual feeding of the OTX
fish in the present study did not seem to be
impaired by OTX, since both sham-operated and
OTX fish gained weight similarly during the one-
month experimental period. It is known in the
goldfish that GnRH stimulates growth hormone
release from the pituitary gland [36]. The results
of present study suggest that GnRH in the TN is
not likely to be involved in the regulation of
growth hormone secretion. Neuromodulatory
effects are suggested as one of physiological func-
tions of TN-GnRH in teleosts [11, 33], but what
aspects of brain activities are modulated by the
TN-GnRH should be elucidated.
Although there are some studies on changes in
brain GnRH contents in relation to gonadal
maturation, a clear correlation between brain
GnRH content and gonadal maturity has not al-
ways been observed [17-22]. Since the present
study suggests that most of sGnRH contents in the
brain reflect those in fibers originating from TN
cell bodies, changes in GnRH contents measured
in those studies may reflect the activities of GnRH
producing-cells in the TN system. In fact in masu
salmon, sGnRH contents of most of discrete brain
areas showed seasonal changes parallel to those of
the olfactory bulbs [18], where cell bodies of
772
sGnRH neurons shown by immunocytochemistry
to be abundant [12]. More recently, Suzuki et al.
[37] revealed by in situ hybridization techniques
that the intensity of hybridization signals were
greater in the olfactory neurons and the olfactory
bulbs than in the hypothalamic area in masu
salmon. These results combined with the present
study suggest that changes in the contents of
GnRH attributed to the TN system seem to mask
changes in the hypothalamic GnRH which are
considred to regulate GTH release. Since the
axonal transport of GnRH from the TN system to
other brain areas can be easily blocked by OTX in
goldfish, OTX fish appear to be good models for
studying relationship between gonadal maturation
and GnRH of hypothalamic origin.
In summary, the present study suggests that
most of the sGnRH in the brain originates from
the TN system, and that this GnRH is not essential
to gonadal maturation in goldfish. The physio-
logical functions of the TN system and sGnRH of
TN origin remain unknown. The origin of brain
cGnRH-II and its functions should also be eluci-
dated. Presently, we are further examining the
involvement of YTN-sGnRH in vitellogenesis,
ovulation, and sex behavior, and using specific
antisera, the distribution of sGnRH and cGnRH-II
neurons are being immunocytochemically studied.
ACKNOWLEDGMENTS
We thank Dr. Yoshitaka Oka, Zoological Institute,
University of Tokyo, for his critical reading of the
manuscript. This study was supported in part by Grants-
in-Aid for Scientific Research from the Ministry of
Education, Science, and Culture and by a Grant-in-Aid
(Bio Media Program 91-II-2-5 from the Ministry of
Agriculture, Forestry, and Fisheries of Japan.
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ZOOLOGICAL SCIENCE 9: 775-784 (1992)
Antagonism of Prolactin and Growth Hormone: Impact on
Seawater Adaptation in Two Salmonids, Salmo trutta
and Oncorhynchus mykiss
STEFFEN S. MADSEN!” and Howarp A. BERN*
‘Institute of Biology, Odense University, Campusvej 55, DK-5230 Odense M,
Denmark; *Department of Integrative Biology, Cancer Research
Laboratory and Bodega Marine Laboratory, University of
California, Berkeley, California 94720, U.S.A.
ABSTRACT—The effect of simultaneously-injected prolactin and growth hormone on short-term
adaptation to seawater was investigated in two salmonid teleosts, Salmo trutta and Oncorhynchus
mykiss. Freshwater-adapted fish were given 5—6 injections of prolactin, growth hormone or combina-
tions of the two hormones on alternate days and subsequently challenged by transfer to seawater. Major
plasma ions, osmolarity, muscle water content and gill Nat, K*-ATPase activity were measured. In
both species, increased plasma ions and decreased muscle water were observed 2-3 days after transfer to
seawater. Growth hormone significantly reduced post-transfer changes in plasma ions and muscle
water, whereas prolactin injections alone had no effect on either freshwater-adapted or seawater-
challenged fish. However, when prolactin was injected simultanously with growth hormone, prolactin
significantly abolished the seawater-adaptive effect of growth hormone in a dose-related manner.
Prolactin also significantly reduced the growth hormone-induced stimulation of gill Na*, K ‘-ATPase
activity. We conclude that prolactin is antagonistic to growth hormone during seawater adaptation and
© 1992 Zoological Society of Japan
development of hypoosmoregulatory mechanisms in salmonids.
INTRODUCTION
Despite a close similarity in the primary struc-
tures of prolactin (PRL) and growth hormone
(GH), there is much evidence to suggest an
antagonistic relationship during development of
hypoosmoregulatory mechanisms in salmonids.
Plasma PRL and GH levels are normally inversely
related during both smoltification [1, 2] and sea-
water (SW) adaptation [1, 3-7], with PRL levels
decreasing and GH levels increasing in both
events.
PRL is essential for freshwater (FW) survival
and osmoregulation in several euryhaline teleosts
[8-10]. The importance of PRL in FW adaptation
of salmonids is, however, less clear, as
hypophysectomized fish survive well in FW with
little or no ionic disequilibrium [11-13]. PRL
causes hypernatremia when injected into FW- and
Accepted April 19, 1992
Received March 7, 1992
SW-adapted teleosts including salmonids (see [10],
[14], [15]). Furthermore, PRL decreases Cl~
excretion by opercular membrane chloride cells
[16] and reduces chloride cell size in seawater-
adapted tilapia, Oreochromis mossambicus [17].
The importance of GH during the early phase of
SW adaptation and in smoltification is well estab-
lished for several salmonids including sea trout,
Salmo trutta, and rainbow trout, Oncorhynchus
mykiss [18-22]. GH has a hypoosmoregulatory
effect, stimulating gill chloride cell proliferation
and Na‘, K*-ATPase activity in vivo [20-22], but
not in vitro [23]. Osmoregulatory effects of GH in
nonsalmonid fishes have not yet been reported.
As the above evidence suggests that GH and
PRL may act antagonistically during SW adapta-
tion, the present study aimed to examine their
interaction following simultaneous injection into
intact fish. The impact of hormone manipulation
was studied for a period before and during the
early phase of SW adaptation. Major plasma ion
levels, muscle water content and gill Na‘, K*-
7716 S. S. MADSEN AND H. A. BERN
ATPase activity were measured.
MATERIALS AND METHODS
Food was withheld from all fish used in the
following experiments beginning two days before
and lasting throughout each experiment. Fish
were briefly anesthetized in 0.01% phenoxyetha-
nol prior to tagging, injection and sampling.
Experiment 1
Fish and protocol Immature sea trout (Salmo
trutta, age 1+, mixed sexes, mean weight 66.1+
1.9g, N=160) were obtained in October 1989
from a commercial trout farm in Egtved, Jutland,
Denmark, where they had been reared under
natural photoperiod and water temperatures. The
fish were brought to the Boegebjerggaard Marine
Laboratory (Odense University), separated into
four groups by fin clipping, and acclimated to the
experimental conditions in fresh water (FW; 10°C,
12:12 L:D artificial photoperiod).
After two weeks of acclimation (=day 0), fish in
the four groups were injected intraperitoneally
according to body weight with saline, ovine growth
hormone (NIADDK-oGH-15: 2 g/g), ovine pro-
lactin (NIADDK-oPRL-19: 2 ug/g) or combined
oGH-+o0PRL (2 ug/g each). Hormones were dis-
solved in saline (0.9% NaCl, 0.5% bovine serum
albumin, pH 9.0) prior to injection, and the injec-
tion volume was 2 yl/g. Hormone doses were
based on those used in previous studies [17, 21,
22].
During the following 14 days, all groups were
given five additional injections with the above
dosages (on days 2, 4, 7, 9 and 11). Three days
after the last injection (day 14), subgroups from
each group were directly transferred to 25 ppt
natural seawater (SW; 10°C), where they were
held for 3 days or until sampling.
Sampling and analyses Subgroups of eight fish
were sampled from each group 3 days after the last
injection in FW, and 2 and 3 days after transfer to
SW. Eight additional fish were sampled from the
control group on day 0 before the first injection.
Blood was drawn from the caudal vessels using
heparinized syringes and centrifuged at 5000Xg;
the plasma was frozen (—20°C) until analysed.
After bleeding, the fish were decapitated, and a
piece of paraxial muscle was dissected and weighed
for determination of muscle water content.
Plasma sodium and potassium were determined
by flame photometry (Instrumentation Laboratory
243), and plasma magnesium and total calcium
analysed by atomic absorption spectrophotometry
(Perkin Elmer 2380, Mountain View, CA).
Plasma chloride was determined by coulometric
titration (Radiometer CMT 10, Copenhagen).
Muscle water content was determined as weight
loss after drying at 105°C until constant weight was
attained (48 hours).
Experiment 2
Fish and protocol Steelhead trout (Oncorhyn-
chus mykiss, age 0+, mixed sexes, mean weight
71.5+1.4 g, N=130) used in this experiment were
obtained from the California Department of Fish
and Game Warm Springs Hatchery in November
1991. They were transported to the Bodega
Marine Laboratory and acclimated in outdoor
raceways for 2 weeks in freshwater prior to experi-
mentation (12-13°C). During that period the fish
were randomly separated into 6 groups and tagged
using visible implants in the clear postocular tissue.
On days 0, 2, 4, 6 and 8, fish in the six groups
were injected intraperitoneally with one of the
following hormone doses/combinations: Con:
saline; GH: 2 ug/g oGH; PRL: 2 ug/g oPRL; GH
+PRLI1: 2 ug/g oGH+0.2 u/g oPRL; GH+
PRL2: 2 ug/g oGH+1 ug/g oPRL; GH+PRL3: 2
ug/g OGH+2 ug/g oPRL. Saline, hormones and
injection volumes were the same as described in
experiment 1.
One day after the last injection (day 9) sub-
groups were transferred directly to 32 ppt natural
SW (12°C) and held for two days.
Sampling and analyses Eight to twelve fish
were sampled from the treatment groups on day 9
in FW and again on day 2 after transfer to SW.
The sampling procedure was the same as in experi-
ment 1, except that gill filaments were also sam-
pled. Filaments were placed in sucrose-EDTA-
imidazole buffer (SEI; 300mM sucrose, 20 mM
EDTA, 50 mM imidazole, pH 7.3), frozen on dry
PRL-GH Antagonism in Salmonid Osmoregulation 7717
ice and stored at —80°C until analyzed for Na‘,
K*-ATPase activity.
Plasma osmolarity was analyzed on a micro
vapor pressure osmometer (Wescor, 5100C,
Logan, UT). Muscle samples were used to deter-
mine muscle water content. Gill filaments were
analyzed for Nat, K*-ATPase activity using a
method slightly modified from McCormick and
Bern [24]. Gill tissue was thawed and homog-
enized in SEI buffer with 0.1% sodium deoxycho-
late and 10 mM mercaptoethanol using a handheld
glass homogenizer. The homogenate was centri-
fuged at 5000 g for 30 sec, and the supernatant
was kept on ice until analysed for Na‘, K*-
ATPase (within 30-60 minutes).
Ten microliters of gill homogenate were added
to 990 yl assay mixture (50 mM imidazole, 1 U/ml
L-lactic dehydrogenase, 2.5 U/ml pyruvate kina-
se, 2mM _ phosphoenolpyruvate, 0.05mM {-
NADH, 0.5mM ATP, 0.4mM KCN, 45 mM
NaCl, 2.5 mM MgCh, 10 mM KCl, pH 7.5). After
mixing, the decrease in absorbance at 340 nm was
recorded over 6 min at 25°C in a Beckman spec-
trophotometer (DU 70; Fullerton, CA). A second
cuvette containing 0.5 mM ouabain was run simul-
taneously, and the difference in rate of NADH
oxidation between assays with or without ouabain
was used to calculate specific Na*, K*-ATPase
activity. Protein content of the gill homogenate
was analyzed according to Lowry et al. [25], and
enzyme activity was expressed as micromoles of
ADP hydrolyzed per mg of protein per hour.
Statistics Statistical differences among groups
were analyzed using CRISP software (CRUNCH
interactive statistical package, San Fransisco, CA).
One-way ANOVA was performed and followed by
Student-Newman-Keuls multicomparison _ test.
Significant differences are reported if the confi-
dence level was >95%.
RESULTS
Experiment 1
The results obtained in Experiment 1 are
summarized in Figure 1. Within each treatment
group, changes in each of the major plasma ions
followed a similar pattern after transfer to SW.
Control, PRL-treated and GH+PRL-treated fish
all showed a major increase in plasma ions on day
2 and a stabilization on day 3. The only significant
difference among these groups was in plasma
Mg** on day 3 in SW (Fig. 1D), when the GH+
PRL-treated group showed a lower value than the
control group. In contrast, the GH-treated fish
showed less deviation from FW values than did all
other treatment groups transferred to SW. In this
group, the posttransfer increase was significantly
less than that seen in controls for plasma Na‘, Cl,
Mg’? and total Ca and less than that in the GH+
PRL-treated for plasma Na* and Cl-. Further-
more, plasma ion levels had returned to FW values
on day 3 after transfer.
Changes in muscle water (Fig. 1F) were inverse-
ly related to changes in plasma ions, with dehydra-
tion taking place on day 2 in SW. GH- and GH+
PRL-treated fish were less dehydrated than con-
trols, with GH-+PRL-treated fish losing more
muscle water than GH-injected fish.
Experiment 2
Plasma osmolarity increased in all groups after
SW transfer (Fig. 2A). However, the magnitude
of the increase varied among the groups and
showed the following order: Con=PRL>GH+
PRL3 >GH+PRL2>GH+PRL1>GH. Changes
in muscle water (Fig. 2B) were inversely related to
changes in plasma osmolarity after SW
transfer and the order of the degree of dehydration
was PRL >Con >GH+ PRL3 >GH+ PRL2>GH
+PRL1I=GH. There was a significant negative
correlation between plasma osmolarity and muscle
water in SW-challenged fish (r7=0.9361, P<
0.01).
The effect of hormone treatment on gill Na‘,
K*-ATPase activity is shown in Figure 3. PRL
significantly decreased and GH significantly in-
creased gill ATPase activity. None of the com-
bined treatment groups showed enzyme activities
significantly different from the control group;
nevertheless, the higher doses of PRL inhibited gill
Na‘, K*-ATPase activity more effectively than
the lower dose.
Regression analyses showed that there was a
significant correlation between pretransfer gill
778
Plasma Nat (mmol/|)
Plasma Cl” (mmol/|)
Plasma Kt (mmol/|)
200
180
160
140
120
100
200
180
160
140
120
100
6.0
5.0
4.0
3.0
2.0
SW transfer
i
|
b
SW transfer
C
oD »
AC
SW transfer
Plasma Mg** (mmol/!)
Plasma Ca (mmol/l)
Muscle water (%)
5.0
4.0
3.0
2.0
1.0
3.0
2.0
1.0
S. S. MADSEN AND H. A. BERN
SW transfer
\ 44
SW transfer
a
—
SW transfer
PRL-GH Antagonism in Salmonid Osmoregulation 779
400 [_] control IN) 2 wg GH + 02 wg PRL/g
2ug GH/g MM 2 ug GH + 1 ug PRL/g
fX] 2 ug PRL/g FA 2 ug GH + 2 ug PRL/g
370
340
310
Osmolarity (mOsm/|)
280
250
Ix<x<h
80
)
(-)
w
~ 79
Cc
~
O°
o 78
®
oO 77
=
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2 76
=>}
=
75 noe
fa OS Xi]
FW groups after 5 injections 48 hr in SW
Fic. 2. Effect of various hormone treatments on plasma osmolarity (A) and muscle water content (B) in O. mykiss in
FW and 48 hr after transfer to SW. Fish were given 5 injections of each dose and combination of PRL and GH in
FW and transferred to 32 ppt SW one day after the last injection. Values are mean+SEM of 8-12 fish. Values
with shared symbols are not significantly different (P<0.05).
Fic.
1. Changes in plasma sodium (A), chloride (B), potassium (C), magnesium (D), and total calcium (E) and in
muscle water content (F) in S. trutta given 6 injections of saline (O), 2 ug/g oRPL (©), 2 ug/g oGH (A) or 2 pg/¢g
oPRL +2 ug/g oGH (@) and subsequently transferred to 25 ppt SW two days after the last injection. Values are
mean+SEM of 8 fish. a, Significantly different (P<0.05) from the control group; b, significantly different from
the GH+PRL-treated group.
780
= L] control
oO 2 ug GH/g
E 8 E] 2 ug PRL/g
O
=
at 6
®
a
ao 4
-
< 7
Mew D
<
=
J
= 0
Fic.
Osmolarity (mOsm/l)
Muscle water content (%)
S. S. MADSEN AND H. A. BERN
a
ist
2 ug GH + 0.2 ug PRL/g
2 ug GH + 1 wg PRL/g
2 ug GH + 2 ug PRL/g
FW groups dea 5 injections
3. Effect of various doses and combinations of PRL and GH on gill Na*
, K*-ATPase activity in O. mykiss.
Fish were given 5 injections in FW and sampled 1 day after the last injection. Values are mean +SEM of 8-10
fish. Values with shared symbols are not significantly different (P>0.05).
400
375
350 Zo
325
79.0
78.0
77.0 ag
76.0
Geri Ctr tae ia TURE Oe Le eel ep anoee aeal
(0) 2.0 4.0 6.0 8.0
Na*,K*-ATPase (umol/mg/hr)
Na‘, K*-ATPase activity and both posttransfer
plasma osmolarity (Fig. 4A: r>=0.7637, P<0.05)
and muscle water content (Fig. 4B: r>=0.6652, P
<0.05).
DISCUSSION
GH treatment was used to induce osmoregula-
tory changes comparable to those expected during
smolting. Both experiments confirmed the SW-
adaptive effect of GH reported earlier for S. trutta
and O. mykiss [19, 21, 22] as well as for other
salmonids [18, 26]. The mechanism responsible
has been shown to involve stimulation of branchial
chloride cell development and Na‘, K*-ATPase
activity in vivo [20-22], but not in vitro [23].
However, more studies are needed to clarify the
exact mechanisms.
The ion-retaining effect of PRL, well established
Fic. 4. Regression analyses of pretransfer gill Na“,
K*-ATPase activity and plasma osmolarity (A) and
muscle water content (B) in O. mykiss 48 hr after
SW transfer. Analyses are based on mean values (+
SEM, N=8-12) from hormone-manipulated fish in
Experiment 2 (see Fig. 2). A: r°-=—0.7637, P<
0.05; B: r= —0.6652, P<0.05.
PRL-GH Antagonism in Salmonid Osmoregulation 781
and developed into a bioassay for PRL bioactivity
in several euryhaline teleosts [27-31], remains
controversial in salmonids [31]. Generally, PRL
seems to play a minor role in FW osmoregulation
in salmonids compared with that seen in other
teleosts. For instance, PRL treatment in the
present study did not have any effect on ionic
balance in FW-acclimated fish, and hypophysecto-
mized (Hx) O. mykiss and O. kisutch survive well
in FW with only minor ion losses [11-13]. Also,
salmonid pituitary PRL secretion in vitro is not
altered by changes in extracellular osmolarity in
the physiological range as occurs in other species
[32, 33]. One study by Oduleye [34], however,
indicated that brown trout depends on pituitary
factors for FW survival.
In contrast to the clear effect of GH, pretreat-
ment with PRL had no effect on iono-osmotic
regulation during the subsequent early phase of
SW adaptation in either species used. By compari-
son, only a minimal hypernatremic effect after 3
injections of 2.5 ug/g chum salmon PRL (sPRL)
into O. mykiss was seen by Bolton et al. ({[19]—see
also Hirano [14]). Any difference in the results
reported could arise from differences in sampling
time, the use of mammalian vs. teleost PRL [29-
31] and size of fish used.
In most salmonids, a hyperosmoregulatory (i.e.,
anti-SW-adaptive) effect becomes evident when
PRL is injected into either SW-adapted fish or into
fish simultaneously injected with GH. Hasegawa
et al. [31] reported that both sPRL (0.5 ug/g) and
oPRL (10 ug/g) induced hypernatremia in O.
mykiss adapted to 50% SW. Similarly, S. Vester-
vang and S. S. Madsen (unpublished) found that
oPRL (2 ug/g) induced hypernatremia and re-
duced muscle water content when injected into O.
mykiss adapted to 28 ppt SW but not when fish
were adapted to 20 ppt SW. Hasegawa et al. [31],
however, did not find any effect of sPRL or oPRL
on ion levels in SW-adapted O. keta fry, suggesting
that either interspecific differences in sensitivity
may exist or that the effect was compensated for by
high levels of SW-adaptive hormones (such as
GH). In our studies, PRL-GH antagonism was
evident when the two hormones were coinjected.
PRL then inhibited the SW-adaptive effect of GH
in a dose-related manner, with the effect of GH
almost abolished when the two hormones were
injected at equal doses. Increasing doses of GH
may antagonize PRL in a similar manner. How-
ever, in a converse experiment conducted at a
different time of year (March instead of Novem-
ber), evidence for the ability of PRL to antagonize
the effect of GH in O. mykiss, although occa-
sionally indicated, could only be considered
equivocal over all (S. S. Madsen, unpublished).
The endocrine effects on hypoosmoregulatory
performance are well correlated with the effects on
gill Nat, K*-ATPase activity (Fig. 5A, B), sug-
gesting a causal relationship. GH stimulated gill
Nat, K*-ATPase in Exp. 2, whereas PRL has a
small but significant inhibitory effect when injected
alone, but in particular when coinjected with GH.
This may be viewed as a “FW-adaptive” effect of
PRL, since lower enzyme levels are normally
associated with FW adaptation in salmonids and
most other teleosts. The effect is in accordance
with a similar inhibitory effect of PRL on gill Na‘,
K*-ATPase in SW-adapted Chelon labrosus [35],
Hx FW-transferred Anguilla japonica [36], Hx
FW-adapted Fundulus heteroclitus [37] and SW-
adapted Morone saxatilis (S. S. Madsen, in prep).
Interestingly, in SW-adapted O. mossambicus,
PRL had no effect on gill Na*, K*-ATPase [17,
38], even though it reduced average chloride cell
size [17] and decreased opercular membrane Cl~
excretion rate [16]. Additional osmoregulatory
effects of PRL that may antagonize SW adaptation
include reduced osmotic permeability of the gill
epithelium [39], reduced intestinal water and ion
transport rates [40], and increased glomerular
filtration rate in vitro in O. mykiss (B. Dunne and
C. Rankin, personal comm.). There are as yet no
reports on the effects of GH on these targets.
During “normal” development of hypo-
osmoregulatory mechanisms (e.g., in smolting
and/or SW adaptation), PRL levels are decreasing
while GH levels are increasing [1—7, 41], suggest-
ing that endogenous release and turnover of PRL
and GH may be inversely controlled during these
processes. Therefore, our results imply that any
development-associated increase in PRL levels
during a hypoosmoregulatory phase of the salmo-
nid life cycle may be critical and incompatible with
hypoosmoregulation due to PRL’s antagonism of
782 S. S. MADSEN AND H. A. BERN
GH. Increased PRL levels reported during sexual
maturation in SW [14, 42-44] could ultimately
cause osmoregulatory maladaptation to SW.
Stress-associated elevations in PRL levels during
smolting [45] may be deleterious to smolt quality
and subsequent performance in SW. Finally, the
effect of experimentally administered hormones
may potentially be antagonized by endogenous
hormones.
The mechanism for the observed antagonism
remains unclear from our in vivo experiments.
Competition between PRL and GH at the receptor
level is unlikely to be a major factor, since dis-
placement studies in various salmonid tissues show
that the GH receptor has only 1-10% of the
affinity for PRL that it has for GH [46-48].
Furthermore, each hormone has its own specific
receptor in several osmoregulatory organs in sal-
monids [36-49]. Although hypothetical at this
moment, it is possible that other endocrine path-
ways may be involved in the observed antagonism.
Insulin-like growth factor-I and “synlactin” are
possible hepatic mediators or synergists for GH
and PRL, according to the somatomedin and “syn-
lactin” hypotheses, respectively [50, 51]. Recent
research in our laboratory has indicated that IGF-I
may stimulate gill Na*, K*-ATPase in O. kisutch
in vitro, thereby supporting the suggestion of
McCormick ef al. [52] that IGF-I is the mediator
for the osmoregulatory action of GH in salmonids.
In conclusion, our study shows that the SW-
adaptive effect of exogenous GH may be abolished
by increasing PRL levels, thus adding another
facet to the multihormonal control of osmoregula-
tion in salmonids.
ACKNOWLEDGMENTS
S.S.M. was a postdoctoral fellow of the Carlsberg
Foundation (Denmark) for part of this study. We are
grateful to Drs. S. D. McCormick and R. S. Nishioka for
their reviews of the manuscript and to H. Ahm
(Odense), A. Corrales and J. S. Endersen (Berkeley) for
their help with the analytical work. NIH and the
National Pituitary Program (Baltimore, U.S.A.) pro-
vided the ovine prolactin and growth hormone used in
this study, and Zenyaku Kogyo Co. of Tokyo provided
research support. This work is a result of research
sponsored in part by NOOA, National Sea Grant Col-
lege Program, Department of Commerce, under grant
number NA89AA-D-SG138, project number R/F-117,
through the California Sea Grant College, and in part by
the California State Resources Agency. The U.S. Gov-
ernment is authorized to reproduce and distribute for
governmental purposes.
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ZOOLOGICAL SCIENCE 9: 785-789 (1992)
© 1992 Zoological Society of Japan
Effects of Ca Concentrations in Culture Medium on the Release of
Calcitonin from Incubated Ultimobranchial Glands
of the Bullfrog, Rana catesbeiana
Hiroyuki Kal-yA, JUNKO OKUYAMA, TAKASHI ISHIJIMA, YUICHI SASAYAMA,
Hipext YosHizAwa! and Cuitaru OGcuro
Department of Biology, Faculty of Science, Toyama University, Toyama 930,
and ‘Department of Oral Anatomy II, Matsumoto Dental College,
Shiojiri, Nagano 399-07, Japan
ABSTRACT—Calcitonin released from ultimobranchial glands incubated in culture media having
different Ca concentrations was determined by rat bioassay. In the first incubation of 30 min, a large
quantity of calcitonin was released into various culture media such as normal medium, and high Ca
media which have 2 times, 3 times and 6 times Ca concentrations compared to the normal medium. In
this term, there was no significant difference in the quantity of calcitonin released into the media among
those groups. However, during 48 hr following the first incubation, in the Ca 2 times medium, calcitonin
quantity released into the medium was maximum.
In the Ca 3 or 6 times medium, the amount of
calcitonin released was less than that of Ca 2 times medium. These results imply that in the bullfrog,
secretion of calcitonin from ultimobranchial glands in vivo may be induced by a suitable rise in the serum
Ca concentration.
INTRODUCTION
Calcitonin is secreted from thyroid glands in
mammals or from ultimobranchial glands in non-
mammals. It has been well known that in mam-
mals, calcitonin is secreted against the rise of blood
Ca levels. In pigs and rabbits, blood calcitonin
level is increased when the thyroid gland is per-
fused with high Ca solution [1-3]. Furthermore,
under in vitro condition, calcitonin secretion from
the thyroid gland of pigs is accelerated by moder-
ate elevation of Ca concentrations in culture
medium [4]. In birds, when the ultimobranchial
gland is perfused with high Ca solution, blood
calcitonin level is increased [5, 6]. Incubated avian
ultimobranchial glands also show the same reac-
tions as in mammals [7, 8]. These facts imply that
in higher vertebrates, the rise of blood Ca level is
one of the factors which accelerate calcitonin
secretion.
On the other hand, it has been reported that in
Accepted May 20, 1992
Received March 16, 1992
bony fishes such as trouts and eels, rise of blood Ca
level does not cause calcitonin secretion from
ultimobranchial glands [9, 10]. Therefore, it is
suggested that mechanisms which trigger calcitonin
secretion may be different between higher verte-
brates and lower ones.
In the present study, effects of Ca concentra-
tions in culture medium on calcitonin release from
incubated ultimobranchial glands of the bullfrog,
Rana catesbeiana, were examined by rat bioassay.
MATERIALS AND METHODS
Male bullfrogs (body weight, 200-300 g) were
purchased from commercial source. One pair of
ultimobranchial glands found near the glottical
sphincter was dissected out carefully under the
binocular microscope, and was immediately put
into an incubation chamber (Lab-Tek 4804, Nunc
Inc.) which contained 1 ml of amphibian Ringer’s
solutions with different Ca concentrations as de-
scribed below. The ultimobranchial glands were
kept at room temperature for 30 min (the first
incubation). Then, the ultimobranchial glands
786
were transferred into a separate chamber which
contained the same kind of incubation medium,
and incubated for 24 hr at 25°C (the second in-
cubation). After that, those were again displaced
into a distinct chamber with the same kind of the
solution, and incubated furthermore for 24 hr at
25°C (the third incubation). Each of the culture
media obtained at each incubation time, which
included released calcitonin, was lyophilized and
frozenly stocked —50°C until use.
Three sets of incubation chambers were pre-
pared according to the incubation period, such as:
30 min (the first incubation), 24 hr (the second
incubation), and next 24 hr (the third incubation).
Each set included four culture media having dif-
ferent Ca concentrations as follows: the normal
amphibian Ringer’s solution, which served as the
standard incubation medium, was composed of
NaCl 6.50 g/l, KCl 0.14g/l, CaCl, 0.12 g/],
NaHCO; 0.20 g/1l, glucose 1 g/l; the other three
were composed of Ringer solutions that contained
2, 3, and 6 times amount of Ca of the standard
medium (Ca 2X, Ca 3X, and Ca 6X incubation
medium, respectively).
Bullfrog calcitonin released from ultimobran-
chial glands into incubation medium was detected
by rat bioassay [11]. Each of the pooled lyophi-
lized samples which were obtained after incubating
3 pairs of ultimobranchial glands separately, was
adjusted to 0.4 ml by saline solution (0.9% NaCl)
and administered to a rat. As a control, the saline
solution was administered. Furthermore, salmon
calcitonin (Novabiochem Inc.) (10, 25 and 100
mU) was administered as a calcitonin standard for
comparison with samples. Blood was samples just
before medium administration and at 0.5, 1, 2, and
3 hr after. Serum Ca concentrations in rats were
determined by atomic absorption spectropho-
tometry (Hitachi-Zeeman 180-70 type). In the
present study, changes in serum Ca levels were
exhibited as decline rates from the initial Ca level.
Furthermore, the areas which were lower than the
initial level were determined to examine the dura-
tion of hypocalcemic effect of the culture medium,
and were exhibited as serum Ca graphs.
Student’s f-test was applied to evaluate the data.
Fic.
H. Kari-ya, J. Okuyama et al.
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S
os TES a 2 3
time(hr)
1. Time courses (0-3 hr) of serum Ca concentra-
tion fall (delta % from the initial levels) in rats after
administrations of the first incubation (30 min)
medium (A) and the second incubation (24 hr)
medium (B) and the third incubation (24 hr)
medium (C). Vertical bar shows mean+SE. Each
symbol means normal Ca medium (4), Ca 2 times
medium (@), Ca 3 times midium (™), Ca 6 times
medium (*) and saline solution (©). The numbers
in parentheses mean number of rats used. Signif-
icantly different from the value of normal Ca
medium: * P<0.05, ** P<0.005, *** P<0.001.
Ca Effect on CT Release in vitro in Frog 787
RESULTS
When incubation media were administered to
rats, serum Ca levels were decreased. Decline
patterns by the first, the second and the third
incubation media having different Ca concentra-
tions are exhibited in Figure 1A, B, C, respective-
ly. In Figure 2, decline patterns are shown when
salmon calcitonin (10, 25 and 100 mU) was admi-
nistered as a calcitonin standard.
In Figure 3A, areas (cm’) declined by the admi-
nistration of normal Ca Ringer (1 x), Ca 2 times
medium (2x), Ca 3 times medium (3X), Ca 6
times medium (6X), salmon calcitonin (sCT: 10,
25 and 100 mU) and saline solution as a control,
which were lower than the initial Ca level during
0-3 hr in each of the incubation time (the first
culture, the second culture and the third culture),
are shown in histograms. Among the areas made
by administration of 4 kinds of the first incubation
media, there was no significant difference, when
153
150 A
100
7 yh
ac
(cm?)
serum Ca 4%
ND
oe)
=e=
Oo
| JA
us (ft
SC OOISNE D) 2
time(hr)
Fic. 2. Time courses (0-3 hr) of serum Ca concentra-
KEE
!
1
1
1
1
1
i}
i]
1
'
1
200
I
I
I
!
!
!
* 1
1
100
|
1
1
i]
if
!
1
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; a !
0
tion fall (delta % from the initial levels) in rats after
administrations of salmon calcitonin 10 mU (@), 25
mU (4) and 100 mU (™). The numbers in parenthe-
ses mean number of rats used. * P<0.05, ** P<
0.01.
B 300
(cm?)
Saline 10 25 100 1X 2X 3X 6X 1X 2X 3X 6X 1X 2X 3X 6X 1X 2X 3X 6X
sCT (ml) Ist culture Qnd culture 3rd culture Total
Fic. 3A. Histograms showing areas (cm7), which are lower than the initial Ca level in Figure 1 and 2, declined by
administrations of saline solution, salmon calcitonin (sCT: 10, 25 and 100 mU), normal Ca Ringer (1x), Ca 2
times medium (2x), Ca 3 times medium (3X) and Ca 6 times medium (6X) in each incubation term of the first
incubation (1st culture), the second incubation (2nd culture) and the third incubation (3rd culture). Significantly
different from 1 x area in each incubation time, but the value was calculated after subtraction of saline area: * P
<0.05, ** P<0.005, *** P<0.001.
Fic. 3B. Histograms showing total areas (cm)), which include all areas during 48.5 hr declined by adiminstrations of
normal Ca Ringer (1x), Ca 2 times medium (2X), Ca 3 times medium (3x) and Ca 6 times medium (6x).
788 H. Kat-ya, J. Okuyama et al.
they were compared after the subtraction of the
area by the saline administration. The calcitonin
released in this incubation time was in large quan-
tities for its short term. Those amounts were
approximately compared to 30-72% of salmon
calcitonin 10 mU judging from the area. However,
among the second incubation media, the area
made by Ca 2X medium was the largest (P< 0.001
to the normal Ringer), which corresponded to
118% of salmon calcitonin 25 mU. The area by Ca
3X medium was also significantly larger than that
by saline control (P<0.05). When the third in-
cubation media were administered, the area made
by Ca 2x medium was also the largest (P<0.005
to the normal Ringer), which was comparable with
87% of salmon calcitonin 25mU. Furthermore,
the areas by Ca 3X and Ca 6X media were also
larger than that by the saline control (P<0.05). In
Figure 3B, the total areas, which include all areas
declined during 48.5 hr, are exhibited in histo-
grams. The area made by Ca 2 medium was 1.9
times larger than the normal Ringer. The area
made by Ca 3X medium was 1.4 times larger than
the normal Ringer. The area by Ca 6X was only
1.1 times larger.
DISCUSSION
It has been reported that in the incubated thy-
roid gland of the pig, secretion of calcitonin into
the medium was concentrated in the first 15 min
period [4]. In the present study, it was known that
also in the bullfrog, release of large quantity of
calcitonin from the incubated ultimobranchial
gland occurred in the first 30 min incubation time.
It has been reported that in the leopard frog, Rana
pipiens, ultimobranchial glands are innervated,
and that the secretion of calcitonin is suppressed
by the nervous system [12]. Therefore, it is
possible that also in the bullfrog, under in vitro
condition, suppressive control of the nervous sys-
tem for ultimobranchial secretion was eliminated.
We reported previously that in the ultimobran-
chialectomized bullfrog tadpoles kept in high Ca
water (20mg/100 ml) for 48 hr, a conspicuous
hypercalcemia was observed [13]. In that experi-
ment, Ca concentration of the environmental wa-
ter was about 3 time that of the serum Ca level (the
average value of the serum Ca before the treat-
ment was 7.4mg/100 ml). Final serum Ca value
was 14.3 mg/100 ml which was 1.9 times that of the
initial value. However, the serum Ca levels of the
sham-operated group did not show any increases.
This fact suggests that in bullfrog tadpoles, the
ultimobranchial glands can secrete calcitonin effec-
tively against at least 2 times rise of the serum Ca
concentrations. Furthermore, when 0.5 ml of ex-
tremely high Ca water (200 mg/100 ml) was in-
fused in the intestine of the ultimobranchialecto-
mized bullfrog tadpoles, the serum Ca concentra-
tions was raised 1.6 times that of the sham-control
group at 24hr later [14]. These observations
suggest that in the ultimobranchialectomized bull-
frog tadpoles, serum Ca level does not elevate
more than twice of the normal level at least for a
short term, even if any treatments are done.
Therefore, also in the adult bullfrog, serum Ca
level may not elevate more than twice of the
normal level. In mammals, it has been known that
secretion rate of calcitonin is directly and linearly
related to the actual increment in plasma Ca level
[1-3]. On the other hand, ultimobranchial glands
of teleosts do not respond to rises of serum Ca
level [9, 10]. In the persent study using adult
bullfrog, it was the Ca 2X medium that was most
effective on releasing calcitonin from incubated
ultimobranchial glands. Therefore, calcitonin
secretion from ultimobranchial glands may be re-
lated to the rise of serum calcium levels, to some
extent in bullfrogs. The Ca 3x medium and Ca 6
x medium were also effective, but less potent than
Ca 2x medium. In anuran amphibians, delicate
control system of calcitonin secretion as in mam-
mals may not have been established.
REFERENCES
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ZOOLOGICAL SCIENCE 9: 791-802 (1992)
© 1992 Zoological Society of Japan
Effects of Chronic Treatment with Chlorpromazine on the Aging
of Hypothalamo-Pituitary-Ovarian Axis in the Rat
Yuta Kopayasut'”, Sencuiro KAwAsHima!, SuMIO TAKAHASHI?
and KatsuMI WAKABAYASHI*
‘Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113,
Department of Pharmacology, Shimane Medical University, Izumo 693,
3Department of Biology, Faculty of Science, Okayama University,
Okayama 700, and *Institute of Endocrinology,
Gunma University, Maebashi 371, Japan
ABSTRACT— Effects of chlorpromazine (CPZ) on the age-related changes of the estrous cycle and the
glial histology in the hypothalamus were studied in female Wistar rats. CPZ (3.0 or 5.5 mg/day/rat) was
orally administered for 6 months beginning at 8 months of age. About 28% of rats given no CPZ
treatment showed prolonged estrus at 13-14 months of age, while about 41% of rats was cycling. In
contrast, none of high-dose CPZ-treated rats of the same age was in a prolonged estrous state and the
difference was statistically significant. The ovarian weight was heavier and the number of Graafian
follicles in the ovary was less in CPZ-treated rats. In the hypothalamic arcuate nucleus, CPZ inhibited
the increase in the number of astrocytic granules. These results indicate that CPZ treatment effectively
delayed the occurrence of some age-related changes of the ovarian function and the hypothalamus in the
rat.
INTRODUCTION
Chlorpromazine (CPZ) is a common antipsycho-
tic drug showing complex biological effects [1].
Although many patients are given CPZ for years,
the reports on the effect of long-term administra-
tion of CPZ on aging processes are a few [2].
Reduction of the accumulation of lipofuscin pig-
ments during aging in neurons of the nucleus
reticularis gigantocellularis by long-term oral
administration of CPZ in C57BL/10 female mice
was observed [3]. In addition, the effect of CPZ on
neurons of rat cerebral cortex in primary culture
was reported by Ohtani and Kawashima [4]. They
observed that CPZ was effective in reducing the
accumulation of lipofuscin pigments in cultured
neurons. The lipofuscin pigments accumulate in
various tissues during aging and they are regarded
Accepted May 28, 1992
Received April 8, 1992
Reprint requests should be addressed to: Dr. S. Kawa-
shima, Zoological Institute, Faculty ef Science, Universi-
ty of Tokyo, Tokyo 113, Japan.
as one of the parameters of aging [5, 6]. These
results led us to propose a working hypothesis that
CPZ affects some aging processes in the brain.
The age-related increase in the astrocytic and
microglial activities in the hypothalamic arcuate
nucleus in female rats was reported [7, 8] and it is
suggested that the age-related increase in the
hypothalamic gliosis may be closely related with
the deterioration of hypothalamic function. Simi-
larly, an increase in the number of reactive micro-
glia (microglia containing more than five granules)
and the number of astrocytic granules in the arcu-
ate nucleus with aging in the rat and mouse was
reported [9].
The decline in reproductive function in female
rats with aging has been observed as the loss of
regular estrous cycles and the manifestation of
irregular cycles, usually characterized by pro-
longed vaginal estrus at middle ages [10-14]. The
age-related deviation of the estrous cycle was
suggested to be dependent on the hypothalamus
(for review, see 13).
The present paper will deal with the effects of
792 Y. KosBAyAsHI, S. KAWASHIMA et al.
chronic administration of CPZ on the occurrence
of prolonged vaginal estrus and the microglial and
astrocytic histology in the arcuate nucleus in
female rats during aging.
MATERIALS AND METHODS
Experimental design
One hundred fifty female rats of the Wistar
strain at 4 weeks of age were purchased from
Nihon Rat Co. Ltd. (Urawa, Japan). Upon arri-
val, they were housed 5 each in an aluminum cage
lined with wood shavings at 22°C under 12 hr-light
(6:00-18:00) and 12hr-dark cycle, and were
given rat chow (CA-1, Clea Japan Inc., Tokyo)
and tap water ad libitum. These animals generally
live to a maximum of 30 months of age. From 31
weeks of age, the commercial chow was replaced
by hand-made chow containing no CPZ. CPZ
administration in hand-made chow began in some
rats at 32 weeks of age. From 56 weeks of age,
commercial chow was supplied again.
The procedures to prepare hand-made chow
were as follows: The chow powder (CA-1) was
kneaded with tap water equivalent in weight to
90% of the chow powder with or without CPZ
(Contomin, Yoshitomi Pharmaceutical Ltd., Osa-
ka) and chopped into cubes of about 15 mmx 15
mm 30mm. The cubes were dried in an oven
about 10 hr at 80°C to 60% of the original weight.
Rats were divided into the following five groups;
(1) High dose CPZ-treated group. Forty rats were
given chow containing 0.35 mg of CPZ per g CA-1
powder. (2) Low dose CPZ-treated group. Forty
rats were given chow containing 0.175 mg CPZ/g.
(3-1) Food restricted group to the level of the food
intake of high-dose CPZ-treated group. Twenty
rats were given the corresponding restricted
amount of hand-made chow containing no CPZ.
(3-2) Food restricted group to the level of the food
intake of low dose CPZ-treated group. Twenty
rats were given the corresponding restricted
amount of hand-made chow containing no CPZ.
(3-3) Control ad libitum feeding group. Hand-
made chow containing no CPZ was given ad
libitum to thirty rats. Food restricted groups were
prepared because the food intake of CPZ-treated
groups was less than that of control group of ad
libitum feeding. The consumption of food per day
in each cage of CPZ-treated groups was recorded
once a week and the same mean daily amount of
chow without CPZ was given to the corresponding
food restricted group in the next week. In high
dose and low dose CPZ-treated groups, the aver-
age uptake of CPZ was 5.5 mg/day/rat and 3.0
mg/day/rat, respectively. During 41st week of
age, water consumption in each cage was mea-
sured daily.
Vaginal smears were recorded during 25-27,
40-42, 53-55 and 60-62 weeks of age daily at
15 :00-17:00.
Animals for plasma hormone determination and
ovarian histology were killed by decapitation at 56,
63 and 68 weeks of age between 11:00 and 14:00.
The plasma was separated from the trunk blood
and kept frozen at —20°C until radioimmunoassay
for luteinizing hormone (LH) and _ prolactin
(PRL). The ovaries and uterus were weighed, and
the ovaries were fixed in Bouin’s fluid.
Histological observation of ovaries
Paraffin sections of the right and left ovaries
were cut at 10 ~m thickness, and one each section
at the first, second and third quarters of the serial
sections, i.e. six sections per animal, was stained
with hematoxylin and eosin. The numbers of
Graafian follicles and corpora lutea larger than 500
ym in diameter were counted.
Histological observation of microglial and astrocy-
tic granules
At 59 weeks of age, five control rats (four
persistent estrous and one cycling rats), five low
dose CPZ-treated rats (one persistent estrous and
four cycling rats) and five high dose CPZ-treated
cycling rats were used for this study. For cycling
rats those at estrus were used. They were anesthe-
tized by i.p. injection of nembutal and perfused
with Lock’s solution from the heart ventricle,
followed by perfusion with 0.12 M phosphate buf-
fer containing 1% glutaraldehyde and 1% para-
formaldehyde. The hypothalami were dissected
out and fixed in 0.1 M cacodylate buffer containing
1% glutaraldehyde and 1% paraformaldehyde for
4hr at 4°C. Then the hypothalami were postfixed
Effects of Chlorpromazine on Rat Aging
in 1% OsO, in the same buffer for 2 hr and
embedded in epon. The ovaries were fixed in
Bouin’s fluid for histological observations.
The microglial and astrocytic changes were
quantified according to the method described by
Brawer et al. [7]. Briefly, coronal semithin sections
1 ym thick were cut at the mid-level of the arcuate
nucleus (4-6 sections). These semithin sections
were stained with toluidin blue and were observed
under a light microscope. The microglia were
identified as relatively small ellipsoidal cells con-
taining toluidin blue-positive granules. The astro-
cytes are small cells generally stained pale with less
frequent granules. Four sections from the ipsi-
lateral arcuate nucleus in each rat were used for
measurements, and the number of reactive micro-
glia (the cell containing more than five granules)
and the number of granules in the astrocyte were
counted.
Radioimmunoassay
Plasma concentrations of LH and PRL in rats at
56 and 63 weeks of age were determined with
radioimmunoassay kits, NIADDK rat LH-I-5,
anti-rat LH S-4, rat LH RP-1 (0.03 NIH unit/mg),
rat PRL-I-3, anti-rat PRL S-6 and rat PRL PR-1
(11 IU/mg) provided by Rat Pituitary Hormone
Distribution Program, NIADDK, National Insti-
tutes of Health, Bethesda, Md. The assay results
for LH and PRL were expressed as mg of NIH-rat-
LH-S, (=NIH unit), and mIU, respectively.
Statistics
The differences in body weight, food intake and
water intake were tested by analysis of variance
(ANOVA). Tissue or organ weights, results of
vaginal smear and blood LH and PRL concentra-
tions were compared by Mann-Whitney’s U-test,
as the distribution of the data did not show appa-
rent “normal distribution”. Concerning the results
of histological observation of the ovaries, the
numbers of follicles and large corpora lutea were
compared by Mann-Whitney’s U-test, and intersti-
tial tissue hypertrophy was compared using
Fisher’s exact probability test as the results were
non-parametric. The numbers of reactive micro-
glia and astrocytic granules were tested by ANO-
VA and Duncan’s multiple range test.
793
RESULTS
Body weight, food intake and water intake
The number of death by 56 weeks of age and/or
apparently unhealthy rats at 56 weeks was as
follows; (1) high dose CPZ-treated group, 4; (2)
low dose CPZ-treated group, 1; (3-1) food res-
tricted group for high dose CPZ group, 0; (3-2)
food restricted group for low dose CPZ group, 3;
(3-3) ad libitum feeding group, 2. There was no
significant difference among groups (Fisher’s exact
probability test).
Changes in body weight during experiments are
shown in Figure 1. The body weight temporarily
decreased in all groups after changing commercial
chow to hand-made chow, and then, it began to
increase. The inhibition of body weight gain in
CPZ-treated groups was dose-dependent (Fig. 1,
Table 1). After the withdrawal of CPZ from the
chow at 56 weeks of age, the rate of body weight
gain recovered to the level of ad libitum feeding
group (Fig. 1). The body weights of food res-
tricted groups were smaller than those of the
corresponding CPZ-treated groups and ad libitum
feeding group, although consumed food amount
450)
++ Ad lib feeding (19)
°-—- CPZlowdose (19)
*-—s €PZ high dose (19)
a
Body weight (g)
300° —_
25 30 35 40 45 50 55 60 65
Age (weeks)
Fic. 1. Changes in body weight by long-term chlorpro-
mazine (CPZ) administration. Hand-made chow
containing 0.175 mg of CPZ per g chow powder was
given to low dose group and that containing 0.35
mg/g, to high dose group of rats from 32 to 55 weeks
of age. The period of CPZ administration is shown
as solid column in abscissa. The numbers in pa-
rentheses indicate the numbers of rats. * P<0.05
compared with the control ad libitum feeding group
(ANOVA).
794 Y. KOBAYASHI, S. KAWASHIMA et al.
TaBLe 1. Effects of chlorpromazine (CPZ) on body weight, food intake and water intake
Caer Body weight Body weight Food intake Water intake
P (g) gain (g) (g/day/rat) (ml/day/rat)
Ad libitum feeding group 398+9 (28) 47 +3 (28) 18.6+0.3 (6) 28.8+1.6 (6)
low dose CPZ group Bw aeY) (39) suiae se (GY) 17.4+0.3* (8) 24.7+0.6 (8)
high dose CPZ group 362+7** (36) 10+3** (36) IS). 7/sE0).3'* (() 19.0+0.6** (8)
Food restricted groups ie
to low dose group 368+8* (17) 8+6** (17) 17.5+0.3* (4) 31.54+2.2* (4)
to high dose group 352+7** (20) 6+3** (20) 16.0+0.3** (4) 28.44+1.17* (4)
Mean+S.E. Numbers in parentheses indicate the number of animals.
Body weight at 55 weeks of age.
Body weight gain during 32 and 55 weeks of age.
Food intake shows the mean during 32 and 55 weeks of age.
Water intake at 41 weeks of age.
* P<0.05, ** P<0.01 compared with ad libitum feeding group (ANOVA).
# P<0.05, ** P<0.01 compared with corresponding CPZ-treated group.
was adjusted to be equal to that of CPZ-treated
groups (Table 1). The difference in body weight
gain between low dose CPZ group and matched
food restricted group was statistically significant
(Table 1).
The food intake in CPZ-treated groups was
significantly and dose-dependently decreased. The
water intake in high dose CPZ-treated group was
significantly less than that in ad libitum feeding
group, while the water intake in low dose CPZ-
treated group and food restricted groups was not
significantly different from that in ad libitum feed-
ing group. Water intake in food restricted groups
was significantly greater than that in matched
CPZ-treated groups.
Estrous cycle, ovarian histology and plasma LH
and PRL concentrations
In the gonadal functions of control rats, there
were no significant differences between ad libitum
feeding group and food restricted groups. There-
Fic. 2. Effects of chlorpromazine (CPZ) adiministra-
tion on frequency distribution of the total number of
proestrous and estrous days (left) and the number of
estrous cycles (right) in young rats at 25-27 weeks of
age (top panel) and rats at 40 to 42 weeks of age
(lower three panels). Arrow indicates the median of
the distribution. n=number of rats. The frequency
distribution of high dose CPZ-treated group was
significantly different from that of the control (P<
0.05) (U-test).
fore, data of ad libitum feeding group and food
restricted groups were combined and served as the
control in the following analyses.
At 25-27 weeks of age, regular 4- or 5-day
estrous cycles were observed (Fig. 2). At 40-42
weeks of age, a few control rats showed prolonged
% Young n=30
Control n=65
CPZ low dose n=39
CPZ high dose n=36
6 2 4 6 8101214161820
135 7 9111315171921
Number of days in
proestrus and estrus
during 3-week period
01234567
Number of estrous
cycles during
3-week period
Effects of Chlorpromazine on Rat Aging
estrus (Fig. 2). Some CPZ-treated rats showed
estrous cycle with relatively long diestrous phase
and a few rats showed prolonged diestrus. The
median of the number of proestrous plus estrous
days of the control group at 42—42 weeks was not
significantly different from those of rats at 25-27
weeks and of high dose CPZ-treated group. The
frequency distribution of prostrous and estrous
days of high dose CPZ-treated group was signi-
ficantly different from that of the control. The
number of estrous cycles during a 3-week period
was about 5 in the young and high dose CPZ-
treated groups and it was about 4 in the control at
40—42 weeks of age (P<0.05, vs. young group).
Fic. 3. Effects of long-term chlorpromazine (CPZ)
administration on frequency distribution of the total
number of proestrous and estrous days (left) and the
number of estrous cycle (right) in rats at 53 to 55
weeks of age. Arrow indicates the median of the
distribution. n=number of rats. The frequency
distribution of the control group was significantly
different from that of high dose CPZ-treated group
(P<0.01) and that of low dose CPZ-treated group
(P<0.02). The frequency distribution of low dose
CPZ-treated group was significantly different from
that of high dose CPZ-treated group (P<0.01). The
frequency distribution of the control group was
significantly different from that at 40 to 42 weeks of
age shown in Fig. 2 (P<0.01) (U-test).
795
The distribution of vaginal smear pattern at 53-
55 weeks of age is shown in Figure3. In the
control group about 28% of rats showed prolonged
estrus, resulting in a mass of distribution locating
toward the right. Another mass was present in the
%
Control n=65
30
u \/
20
10
0
CPZlowdose n=39 v
30 y 1|
20
10
CPZ high dose n=36
Vv
0
0 2 4 6 810121416 18 20 01234567
135 7 9111315171921
Number of days in
proestrus and estrus
during 3-week period
Number of estrous
cycles during
3-week period
TaBLE2. Effects of chlorpromazine (CPZ) administration (from 32 weeks of age to 55 weeks of age) on
some organ weights and plasma LH and prolactin (PRL) concentrations at 56 weeks of age (upper part),
and changes in these parameters at 63 weeks of age, 7 weeks after the cessation of CPZ treatment (lower
part)
Control low dose CPZ group high dose CPZ group
At 56 weeks of age
Ovarian weight (mg/100 g BW) 2.8) 38 IY) (18) A oY SEAS) (10) 28.5+1.0* (9)
Uterine weight (mg/100 g BW) 191+19 (18) 152+ 16 (10) 146+ 13 (9)
LH concentration (mg S,/ml) 0.88 + 0.33 (18) 0.39+0.06 (10) 0.51+0.11 (9)
PRL concentration (mIU/ml) 3.22 +0.99 (18) 1.96 +0.66 (10) 2.49 + 0.74 (9)
At 63 weeks of age
Ovarian weight (mg/100 g BW) WD) ae MF (19) 23.2+2.4% (10) ipo rial (10)
Uterine weight (mg/100 g BW) 192+ 14 (19) 185+ 12 (10) 173+10 (10)
LH concentration (mg S,/ml) 0.52+0.11 (19) 0.44 +0.09 (10) 0.57+1.10 (10)
PRL concentration (mIU/ml) 3.32 £0.66 (19) 2.15+0.46 (10) 1.80+0.28 (10)
Mean+S.E. BW; body weight.
Numbers in parentheses indicate the number of rats.
* P<0.05; compared with control group, * P<0.05, compared with the values at 56 weeks of age (U-test).
796 Y. KoBAYASHI, S. KAWASHIMA et al.
middle showing cyclic estrous pattern (about
41%). The number of estrous cycles of the control
group showed a large variation indicating irregular
cycle of some animals. In contrast, many rats
showed cyclic estrous pattern with a fragmentary
mass at the right in low dose CPZ-treated group.
In high dose CPZ-treated group, the median of
distribution of proestrous and estrous days was
located in the middle with a small mass at the left
showing a prolonged diestrous phase. The fre-
quency distribution was significantly different
among the three groups. The frequency distribu-
tion of the control group at 53-55 weeks of age was
significantly different from that at 40-42 week of
age (Fig.3 vs. Fig.2). The difference in the
frequency distribution of CPZ-treated groups be-
tween 40-42 and 53-55 weeks was statistically not
significant.
Some organ weights and plasma levels of LH
and PRL in three groups at 56 weeks of age are
shown in Table 2. The ovarian weight was signi-
ficantly heavier in high dose CPZ-treated group
than the control group. The low dose CPZ-treated
group showed the same tendency. The uterine
weight was lighter, and LH and PRL levels were
lower in CPZ-treated groups than the control
group, although these differences were statistically
not significant.
TaBLeE 3. Summary of histological observations of the ovaries of rats given
chlorpromazine (CPZ)
Follicles
ale Number of follicles (>500 ~m) Total nue
0-2.0 2.1-4.0 4.1-7.0 CLES
Control ay) 16 6 26
CPZ low 10 7 3 20
CPZ high** 10 4 3 17
Corpora lutea
Number of corpora lutea (>500 um)
Total number
Group ¥ n sre of rats
Control 5 3 18 26
CPZ low y, 0 18 20
CPZ high 0 16 17
Interstitial tissue
Interstitial tissue hypertrophy
Total number
Group 2 ak a of rats
Control 15 3 8 26
CPZ low 13 5 2 20
CPZ high 14 2 1 17
*) Number of rats.
observation of hypothalamus)
Follicles
Ovaries were obtained at 56 weeks (Table 2) and at 59 weeks (for the
Number of follicles per section is shown.
** P<0.01 compared with the control by U-test.
Corpora (—) indicates no C.L. in 6 sections.
lutea (C.L.) (+) indicates less than one C.L. per section.
(+) indicates more than one C.L. per section.
Interstitial (—) indicates no hypertrophy.
tissues (+) indicates slight hypertrophy.
(+) indicates marked hypertrophy.
Effects of Chlorpromazine on Rat Aging 797
When control rats were divided into subgroups,
one showing prolonged estrus (more than 18 days
were in estrus or proestrus out of 21 days) and the
other showing regular estrous cycles (8-13 days of
estrus or proestrus out of 21 days), the ovarian
weight of the former was significantly lighter than
the latter (prolonged estrus, 15.2+2.2 mg/100 g
body weight, n=7; regular cycle, 28.2+2.1 mg/
100 g body weight, n=7; P<0.01). The uterine
weight was significantly heavier (prolonged estrus,
226+23 mg/100 g body weight; regular cycle, 126
+17 mg/100 g body weight; P<0.01) in prolonged
estrus group than regular cycle group. The LH
level was greater in the former group (prolonged
estrus, 1.61+0.80 mg S,/ml; regular cycle, less
than detectable level of 0.25 mg S;/ml; P<0.01 by
Fisher’s exact probability test). The PRL level was
greater (prolonged estrus, 5.6+2.3 mIU/ml; regu-
lar cycle, 1.5+0.5 mIU/ml) in prolonged estrus
group than regular cycle group, however, the
difference was not significant. In CPZ-treated rat
subgroups, the results were similar to those in the
control subgroups.
The ovaries of rats showing regular estrous
cycles in three groups contained some follicles and
corpora lutea, and the hypertrophy of interstitial
tissue was not observed in these ovaries. On the
contrary, the ovaries of rats showing prolonged
estrus in the three groups contained numerous
follicles but no or few large corpora lutea, and
showed the hypertrophy of interstitial tissue. Sum-
mary of ovarian histology at 56 and 59 weeks of
age is shown in Table 3. In the ovaries of 85% of
control rats, more than two large follicles per
section were observed. In the ovaries of 59% of
high dose CPZ-treated group rats and 50% of low
dose CPZ-treated rats, less than two follicles per
section were present. The difference in the num-
ber of follicles between the control and high dose
CPZ-treated group was statistically significant. On
the other hand, less than one large corpora lutea
per section was observed in the ovaries of about
30% of control rats, and in those ovaries the
hypertrophy of interstitial tissue was apparent. In
94% of the ovaries of high dose CPZ-treated rats
and 90% of them of low dose CPZ-treated rats,
more than one large corpora lutea per section was
present. Difference in the number of large corpora
lutea was not statistically significant.
Seven weeks after the cessation of CPZ treat-
ment the same parameters were studied. The
occurrence of estrous and proestrous days during a
3-week vaginal smear test at 60-62 weeks of age is
shown in Figure 4. In CPZ-treated groups, the
incidence of rats showing prolonged estrus became
greater as compared with that at 53-55 weeks of
age, and there was no significant difference from
the value of the control group. The shift of the
frequency distribution was significant in the high
and low dose CPZ-treated groups at 60-62 weeks
of age compared with that of the same animals at
%
Control n=39 fo
y
}
10
0 —
CPZlowdose n=19 |
30 | i?
20} 1
CPZ highdose n=19
30 | ' ' | Vi
20 |
10 j
0
0 2 4 6 8101214161820
135 7 9111315171921
Number of days in
proestrus and estrus
during 3-week period
01234567
Number of estrous
cycles during
3-week period
Fic. 4. Effects of the cessation of chlorpromazine
(CPZ) administration on frequency distribution of
the total number of proestrous and estrous days
(left) and the number of estrous cycle (right) in rats
at 60 to 62 weeks of age. Solid arrow indicates the
median of the distribution. n=number of rats. The
difference in the frequency distribution of the con-
trol group and CPZ-treated group was not statisti-
cally significant. The median of the frequency
distribution of high and low dose CPZ-treated
groups became significantly larger than those
(broken arrows) of the identical groups at 53 to 55
weeks of age (P<0.01) (U-test).
798
53-55 weeks of age.
The ovarian weight of CPZ-treated rats at 63
weeks of age was not significantly different from
that of the control (Table 2). The ovaries reduced
in weight seven weeks after the cessation of CPZ
treatment compared with that during CPZ treat-
ment in low dose CPZ-treated group (Table 2).
Plasma levels of LH and PRL at 63 weeks of age
were similar to those at 56 weeks of age (Table 2).
Observations of ovarian histology revealed no
significant differences among the three groups at
63 weeks of age, although the number of follicles
in high dose CPZ-treated group tended to be
smaller than the other two groups (Table 4). The
increase in the number of follicles in low dose
CPZ-treated group and the increase in the inci-
dence of interstitial tissue hypertrophy in high dose
CPZ-treated group were significant after the cessa-
Y. KoBAyYAsHI, S. KAWASHIMA et al.
tion of CPZ treatment compared with that during
treatment (Tables 3 and 4).
Vaginal smear pattern, organ weights and ova-
rian histology in rats killed at 68 weeks of age (9
high dose CPZ-treated, 9 low dose CPZ-treated
and 20 control rats; data not shown) were similar
among the three groups and were almost the same
compared with those at 63 weeks.
The number of reactive microglia and astrocytic
granules
Figure 5 shows an example of reactive microglia
and astrocytic granules in the hypothalamic arcu-
ate nucleus of a control rat. The effects of CPZ on
the arcuate glial histology are presented in Table
5. In the number of reactive microglia, no signi-
ficant differences were detected among the three
groups, although the number of reactive microglia
TABLE 4. Summary of histological observations of the ovaries of rats 7 weeks after
the cessation of chlorpromazine (CPZ) treatment
Follicles
Gat Number of follicles (>500 «m) Total Aono
0-2.0 2.1-4.0 4.1-7.0 of rats
Control 6» 7 6 19
CPZ low* 2 4 ti
CPZ high A i rm
Corpora lutea
Number of corpora lutea (>500 «m)
Total number
GrONp a u ie of rats
Control 1 3 15 19
CPZ low 1 8 10
CPZ high 0 2 8 10
Interstitial tissue
Interstitial tissue hypertrophy Totalbwumbee
Group i of rats
= ae +
Control 12 2 5) 19
CPZ low 4 4 2 10
CPZ high* 4 3 3 10
@) Number of rats.
# P<0.05, compared with the score during CPZ administration (Table 3) by U-test
(follicles) or Fisher’s exact probability test (interstitial tissue).
Follicles; Number of follicles in a section is shown.
interstitial tissue, refer to Table 3.
For indices of corpora lutea and
Effects of Chlorpromazine on Rat Aging 799
Fic. 5. Mid-level of hypothalamic arcuate nucleus of a
control rat. Toluidin blue preparation. Microglia
(arrows) and astrocytic granules (arrow heads) are
visible. Lipofuscin-like granules are present in the
neurons (N). Scale: 5 um.
TaBLE5. Glial histology in the hypothalamic arcu-
ate nucleus in chlorpromazine (CPZ)-treated rats
killed at 59 weeks of age
Number Number of Number of
Group of reactive astrocytic
rats microglia® granules
Control 5 7.6+1.1 SERS Ie
CPZ low 5) 7.0+1.9 ADS seas
CPZ high 5 3.2+0.6 Aide
Mean+S.E.
a; Number of microglia containing more than five
granules in four semithin sections.
b; Total number of granules in astrocytes in four
sections.
** P<(0.01, among groups (ANOVA).
## P<(.01, difference from the control (Duncan’s
multiple range test).
tended to decrease in high dose CPZ-treated
group. On the other hand, the ANOVA clearly
showed a singificant effect of CPZ treatment on
the number of astrocytic granules. The numbers of
astrocytic granules in low and high dose CPZ-
treated groups were significantly less than that in
the controls (P<0.01 in both comparisons).
DISCUSSION
Reduction in body weight gain by chronic CPZ
treatment in rats in the present study agrees with
the previous report using mice [3]. A significant
decreases in food intake may be the main reason
for the reduction in the rate of body weight
increase. Although food intake was manipulated
to be the same by food restriction, body weight
gain was significantly less in food restricted control
rats than CPZ-treated rats. We have already
reported that 5 months treatment with high dose of
CPZ reduced the ambulatory activity in open field
test in 13-month-old rats [15]. Such a change in
behavior in CPZ-treated rats may correlate to the
energy metabolism and may result in larger body
weight gain in CPZ-treated groups than food in-
take-matched control rats.
Food restriction increases longevity [16-18].
This is the reason why we prepared a food res-
tricted group for each dose group. However, there
was no difference between ad libitum feeding
group and food restricted groups as concerns the
parameters of gonadal function in the present
study. Therefore, it is considered that the effects
of CPZ on gonadal function observed in the pre-
sent study were not the consequences of the reduc-
tion in food intake and body weight gain. Previous
studies restricted food to 40 to 60% of ad libitum
intake [17-18]. In the present study, the reduction
of food intake by low and high doses CPZ-treated
groups were to 94 and 84% of ad libitum intake,
respectively. Such a small reduction of food intake
may have no influence on aging.
Samorajski and Rolsten [3] could not detect any
change in water intake by CPZ treatment. The
present results disagree with theirs. On the other
hand, water intake of food restricted groups was
almost the same as that of ad libitum feeding
group, indicating that the reduction in water intake
by CPZ treatment is mediated not through the
reduction of food intake.
Vaginal smear records showed that the adminis-
tration of CPZ was effective for the maintenance
of regular estrous cycle, when prolonged estrous
State was predominant in age-matched control
rats. Ovarian weight and the number of follicles in
high dose CPZ-treated rats were significantly diffe-
rent from those in the control, due to the differ-
ence in the proportion of subtypes of gonadal
function between the two groups. Any differences
on some other organ weights but ovaries and on
ovarian histology were not observed between
CPZ-treated rats showing estrous cycle and control
800 Y. Kopayasui, S. KAWASHIMA et al.
cyclic rats, indicating normal gonadal function in
the cyclic CPZ-treated rats. The blood hormone
levels, uterine weight and ovarian histology did not
conflict with this interpretation. At 63 weeks of
age, 7 weeks after the cessation of CPZ adminis-
tration, regular estrous cycles disappeared in most
CPZ-treated rats.
In our separate study, high dose of CPZ chow
was administered for 10 days to 12-month-old rats
showing persistent estrus, but any recovery to
regular estrous cycles was not observed (unpub-
lished observation). The maintenance of estrous
cycle by CPZ seems to be related with long-term
administration of this agent from younger ages.
Aschheim [12, 19] demonstrated that the trans-
plantation of ovaries from young rats into ovar-
iectomized aged animals which had been in persis-
tent estrous state did not result in reinitiation of
estrous cycles, and he suggested that the origin for
the loss of estrous cycle in aged rats was not at the
ovarian level. Electrical stimulation of the preop-
tic area of aged rats restored resproductive func-
tion [20]. Maintenance of cyclic cornification of
the vaginal epithelium was observed in young adult
female rats given heterochronic transplantation of
the pituitary and the ovary from aged female rats.
These results indicate that the age-related devia-
tion of the estrous cycle was not dependent on the
ovary but on the hypothalamus.
The hypothalamus or higher central nervous
system is important as the action site of CPZ in the
present study. CPZ treatment decreased the accu-
mulation of astrocytic granules compared with
controls. The increase in the glial activities in
microglia and astrocytes with aging was reported
[9]. In a preliminary study, we also found the
age-related increase in glial activities. In Wistar/
Tw strain maintained in our laboratories, the num-
ber of reactive microglia in the arcuate nucleus in
19-21-month-old females showing persistent
estrus (33.8+3.5 cells/4 sections, n=6) was signi-
ficantly greater (P<0.001) than that in 5-month-
old females at estrus (5.6+0.3 cells/4 sections, n=
5). Similarly, the number of astrocytic qranules in
the arcuate nucleus in aged females (871+43
granules/4 sections, n=7) is significantly higher (P
<0.001) than that in young females (215+25
granules/4 sections, n=5). Similar changes were
reported in other brain areas: for example, the
increase in the numerical density of hypertrophied
astrocytes in the hippocampus in rats with aging
[22]. The reduction in astrocytic granules by CPZ
treatment in the present study may have some
relevance to the delay in gonadal aging.
CPZ treatment reduced the accumulation of
lipofuscin pigments in the nucleus reticularis
giagantocellularis of mice [3]. They interpreted
the effect of CPZ on its action on the biomem-
brane properties. It is suggested that the forma-
tion of lipofuscin was related with peroxidation of
lipids in biomembranes [6]. In support of this
view, some anti-oxidants or free-radical scavengers
have been reported to reduce the accumulation of
lipofuscin pigments and show other anti-aging acti-
vities [23-26]. The anti-oxidative activity or
effects of CPZ on biomembrane properties may
have some relation with the present anti-aging
effects of CPZ.
Acute administration of CPZ (25 mg/kg body
weight) stimulates PRL release from the pituitary
due to its action as a dopamine antagonist [1, 27—
29]. Inhibition of gonadal function by enhanced
PRL release and the occurrence of some pro-
longed diestrous rats in CPZ-treated group may be
correlated with the PRL release activity of CPZ.
However, in the present study, blood PRL level
was somewhat lower in CPZ-treated rats at 56
weeks of age. Measurement of blood PRL level in
CPZ-treated rats at earlier period, especially dur-
ing prolonged diestrous state, was necessary to
clarify the correlation. The possibility that other
effects of CPZ, such as a non-competitive acetyl-
choline blocking action [30] on the central nervous
system, caused the delay of aging in the hypothala-
mo-hypophysial-gonadal system cannot be ex-
cluded.
To conclude, delay of the occurrence of some
age-related changes of the ovarian function and
the hypothalamic glial histology by chronic CPZ
treatment in the rat was evident.
ACKNOWLEDGMENTS
The authors wish to express their gratitudes to Dr. S.
Raiti and the Pituitary Hormone Distribution Program,
the National Institute of Arthritis, Diabetes and Diges-
Effects of Chlorpromazine on Rat Aging
tive and Kidney Diseases (NIADDK) and Dr. A. F.
Parlow, Pituitary Hormones/ Antisera Center, Harbor-
UCLA Medical Center for kind supply of RIA kits. This
work was supported in part by Grants-in-Aid from the
Ministry of Education, Science and Culture, Japan (Nos.
63304008, 02404007).
10
11
1,
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ZOOLOGICAL SCIENCE 9: 803-809 (1992)
Differential Effects of Testosterone and 5a-Dihydrotestosterone
on Growth in Mouse Submandibular Gland
KAZUHIKO SAWADA and TETSuO NOUMURA
Department of Regulation Biology, Faculty of Science,
Saitama University, Urawa, Japan
ABSTRACT—Testosterone and 5a-dihydrotestosterone (DHT) were compared their morphometrical
effects on the mouse submandibular gland in relation to the developmental profiles of their serum levels.
In male mice, serum levels of both testosterone and DHT were dramatically increased between days 20
and 30, when sexual difference of the submandibular gland occurred morphometrically. In 30-day-old
males, serum testosterone and DHT levels were 2.9+0.59 ng/ml and 1.0+0.21 ng/ml, respectively
(3:1 in the ratio). Daily injection of testosterone and DHT for 1-20 days starting on day 20 induced
morphometrical changes in the glands of neonatally-castrated mice. The glandular weight gain was
caused only in males by treatment with testosterone for 10-20 days, but in both sexes by DHT for 7-20
days. The relative occupied area (ROA) of the granular convoluted tubules (GCT) in castrated mice of
both sexes were increased by treatment with testosterone for 10-20 days, but by DHT for 7-20 days.
The mitotic rates of GCT were increased only in males by treatment with testosterone for 10-13 days,
but in both sexes by DHT for 2-7 days.
The present study suggests that androgenic effects of these two androgens on growth in the mouse
submandibular gland are different, being more affected by DHT than by testosterone, and also that
© 1992 Zoological Society of Japan
androgen responsiveness of the gland is sexually different, being higher in male than in female.
INTRODUCTION
In rodents, the male submandibular gland is
larger and has more complex morphology than the
female one. The glandular contents of biologically
active polypeptides including nerve growth factor,
epidermal growth factor, renin and proteases, are
higher in the male than in the female, being
responsive to androgens [1-7]. By histological,
ultrastructural and morphometrical studies, both
sexes in the mice experience a similar mor-
phogenesis of the mouse gland during perinatal
development, and then the sexual difference arises
at 3-4 weeks of age, when the granular convoluted
tubules (GCT) grows more rapidly in the male
than in the female [8-11]. The masculine develop-
ment of GCT in the gland is caused by androgens,
testosterone and 5a-dihydrotestosterone (DHT),
but not by estradiol-17 [11]. In a completely
androgen-independent state (neonatally-castration
Accepted June 2, 1992
Received April 8, 1992
and androgen-insensitive Tfm mutation), the gland
displays the feminine development [11, 12].
The present study was aimed to compare the
effects of testosterone and DHT on morphometric-
al changes in the mouse submandibular gland in
relation to the developmental profiles of their
circulating levels.
MATERIALS AND METHODS
Animals
CD-1 mice were obtained from Charles River
Japan Co. and maintained by randomly mating in
our laboratory. The animals were given a com-
mercial diet (CRF-1: Charles River Japan Co.)
and tap water ad libitum and were kept at 23+1°C
under 12 hr artificial illumination (from 8:00 to
20:00).
Hormone assay
In mice of both sexes on days 20, 30 and 90,
blood sample was collected from the tail vein
804 K. SAWADA AND T. Noumura
under the ether anesthesia. The serum was sepa-
rated and stored at —20°C until assayed. Serum
levels of testosterone and 5a-dihydrotestosterone
were separately determined by a radioimmunoas-
say. The inter- and intraassay variations of testos-
terone were 11.5% and 5.2%, and those of DHT
were 12.4% and 5%, respectively. Individual
values were the mean of the duplicate determina-
tions and were expressed as nanogram per millili-
ter of serum.
Treatments of animals and histological procudure
Both male and female mice were castrated on
day 0 and were subcutaneously given daily treat-
ments with testosterone (10 or 100 ug), DHT (10
or 100 ug) or sesame oil vehicle alone (0.1 ml) for
1, 2, 4, 7, 10, 13, 16 and 20 days starting on day 20.
Mice were killed on days 21, 22, 24, 27, 30, 33 36
and 40, respectively. All animals were sub-
cutaneously given a single injection of colchicine (5
pg/g body weight) dissolved in 0.9% NaCl, 5 hr
before they were killed to arrest cell division at
metaphase. Submandibular glands were weight
and fixed in Bouin’s solution, embedded in par-
affin and sectioned at 8 wm. Sections were stained
with Delafield’s hematoxylin and eosin.
Morphometrical Methods
The sectional figures of submandibular glands in
the microscopic enlargements (x 400) were traced
by the camera lucida, and the areas cf GCT were
measured by the picture analyzer (Logitec K-510,
Kantou Denshi Co., Japan) connected with a
microcomputer (NEC PC-9801DA). The areas
were expressed as percentages of total area (the
relative occupied area, ROA). Cell height of GCT
was measured in randomly chosen five sections.
Mitotic Activities
Cells in division per 5S00-1,000 cells were
counted in the GCT of the glands. Mitotic rate was
estimated by counting the cell at metaphase per 5
hr in 100 cells.
Statistical Analysis
Data were statistically analyzed by Student’s
t-test or by Cochran-Cox test.
RESULTS
Serum levels of testosterone and DHT in mice
On day 20, serum testosterone levels showed no
sexual difference statistically, but were slightly
higher in males than in females (0.35 +0.08 ng/ml
and 0.23+0.01 ng/ml). In male mice, serum tes-
tosterone levels dramatically raised on day 30 and
attained adult levels (Fig. 1A). Similarly, serum
DHT levels were slightly higher in males than in
females on day 20 (0.45+0.10 ng/ml and 0.11+
0.01 ng/ml) and then the DHT levels in males
dramatically increased to adult levels by day 30
(Fig. 1B). At 30 and 90 days of ages, serum levels
of testosterone were 3-fold higher in males and
2-fold higher in females than those of DHT, re-
spectively (Fig. 1A and 1B).
testosterone A
serum levels (ng/ml)
~~ IN) (et)
ag
>
20 30 90
age in days
Fic. 1. Serum levels of testosterone and DHT in mice.
a: P<0.05, b: P<0.01 vs age-matched females
(Cochran-Cox test).
Androgens and Mouse Submandibular Gland 805
male A female
150 testosterone
e oil
©1049
ia} 10049
100
50
weight of the glands (mg)
0) 10 20 0 10 20
treatment periods (days)
Fic. 2. Change in the weight of the submandibular glands of neonatally-castrated mice, treated with testosterone (A,
C) and with DHT (B, D) for 1-20 days from day 20. a: P<0.05, b: P<0.02, c: P<0.01, d: P<0.002, e: P<0.001
(Student’s ¢-test).
male i female
60
testosterone c
@ oil
° 10u9
40; © 100yu9
~
°
20
~—
i—
©
©
—
S D
<x
ie)
x
ro) 10 20 0 10 20
treatment periods (days)
Fic. 3. Change in the relative occupied areas (ROA) of GCT of the submandibular glands of neonatally-castrated
mice, treated with testosterone (A, C) and with DHT (B, D) for 1-20 days from day 20.
806 K. SAWADA AND T. NOUMURA
Changes in the mouse submandibular gland after
treatments with testosterone and DHT
As circulating levels of androgens, both testos-
terone and DHT, showed a dramatically increase
between days 20 and 30, we compared the effects
of these androgens on morphometrical changes in
the submandibular glands of neonatally-castrated
mice for 20 days from day 20. The gland weight in
castrated males gradually increased and became
significantly higher after 7 days of treatment with
testosterone (Fig. 2A). In castrated females, the
gland weight gain was ultimately induced after 16
days by treatment with 100 ug testosterone, but
was failed by 10 ug testosterone during the period
of 20-day treatment (Fig. 2C). Seven days and
longer treatment with DHT caused to increase
significantly the gland weight in both sexes. DHT
was more effective on the gain in gland weight than
testosterone (Fig. 2B and 2D).
In castrated males, the ROA of GCT were
male
25
fF testosterone
height of GCT cells (um)
gradually increased and reached plateau after 13—
16 days by treatment with 10-100 ug testosterone
or 10 wg DHT, and after 10 days by 100 ng DHT
(Fig. 3A-D). Similarly, the height of GCT cells in
both sexes significantly increased after 7 days by
treatment with testosterone and after 4 days by
DHT (Fig. 4A-D). These androgenic effects were
dose-dependent, and DHT was more effective on
the GCT stimulation than testosterone (Fig. 3 and
4). Testosterone and DHT had no effect on the
ROA of the intercalated ducts (ID) and excretory
striated ducts (SD), and both androgens did not
caused any changes in the size of acini in the glands
(data not shown).
The mitotic rates of GCT in castrated males
were significantly increased by treatment with 10-
100 4g testosterone and with 10 “7g DHT only after
10-13 days (Fig.SA and 5B). In castrated
females, 10-100 ug testosterone and 10 ~.g DHT
failed to increase the mitotic rates during treat-
ment periods of 20 days (Fig. 5C and 5D). By
female
20 0 10 20
treatment periods (days)
Fic. 4. Change in the height of GCT cells of the submandibular glands of neonatally castrated mice, treated with
testosterone (A, C) and with DHT (C, D) for 1-20 days from day 20. a: P<0.05, b: P<0.005, c: P<0.001
(Student’s t-test), d: P<0.001 (Cochran-Cox test).
Androgens and Mouse Submandibular Gland 807
male
one testosterone
e ol/
fe) 109
mitotic rate of GCT cells (%)
female
C
D
e
d
c
(6) 10 20
treatment periods (days)
Fic. 5.
Change in the mitotic rates of GCT of the submandibular glands of neonatally-castrated mice, treated with
testosterone (A, C) and with DHT (B, D) for 1-20 days from day 20. a: P<0.05, b: P<0.01, c: P<0.01, d: P<
0.005 (Student’s t-test), e: P<0.05, f: P<0.01 (Cochran-Cox test).
treatment with 100 ~g DHT, however, the mitotic
rates were significantly increased after 2 days,
reached a peak after 4 days, and then gradually
decreased to the control levels after 10 days (Fig.
5B and 5D). Neither testosterone nor DHT had
influence on the mitotic rates of the other three
regions in the glands (data not shown).
DISCUSSION
In adult mice, the submandibular gland consists
of acini, ID, GCT and SD. Among these struc-
tures, the GCT develops more in the male than in
the female. The mouse gland has been studied
histologically, ultrastructurally and morphometri-
cally during the pre- and postnatal development.
Both sexes experience a similar morphogenesis of
the gland during early postnatal development, and
then the sexual differences occur at 3-4 weeks of
age, when the GCT grows more rapidly in the male
than in the female [8-11]. The mouse subman-
dibular gland contains both the cytosolic and the
nuclear androgen receptor [13-17]. The cytosolic
androgen receptor in the glands of male mice
increases during postnatal development and
attains adult levels by day 20 [13, 16], while
circulating testosterone levels begin to rise on day
20 [16]. In the present study, serum levels of
testosterone and DHT dramatically increased be-
tween days 20 and 30 (Fig. 1A and 1B). Adminis-
tration of both androgens to neonatally-castrated
mice for 20 days from day 20 caused to increase the
gland weight and the ROA of GCT, the cell height
of GCT and the mitotic rates of GCT, DHT being
more effective than testosterone (Fig. 2-5). In our
previous study, estradiol-178 did not induce any
morphometrical changes in the mouse gland [11].
These results suggest that the masculine develop-
808
ment of the mouse gland is attributed to an in-
crease in the circulating androgen levels during
development. In the mouse gland, testosterone
acts in its own right, but not as the precursor of
DHT or other biologically active metabolites, be-
cause it is not converted in vitro to its metabolic
derivatives by the glands from 20- and 30-day-old
mice (Sawada and Noumura, unpublished
observations). In our male mice at 30 days of ages,
circulating DHT levels were 1.0+0.21 ng/ml and
were about 35% of the testosterone levels (Fig.
1B). In comparison with male rats at puberty
reported [18], the DHT levels were approximately
4-fold over and the serum DHT/ testosterone ratio
was approximately 2-fold higher in our male mice.
Therefore, both testosterone and DHT may act
independently on the masculine development of
the mouse gland, but it is not yet solved which is a
predominant androgen. Research is required to
examine further the properties of the androgen
receptor of the gland by using both androgens as
ligands.
In our previous study, administration of 100 ng
DHT for 10 days from day 20 failed to increase the
mitotic rates of GCT in neonatally castrated mice
of both sexes [11]. In the present study, however,
daily 100 ng DHT increased the mitotic rates of
GCT between 2-7 days only, indicating a peak at 4
days, and then gradually declined to the control
levels at 10 days (Fig. 5B and 5D). Therefore, it
turned out that the observation after 10 days was
not suitable for examining the effects of DHT on
the mitotic activity in our previous study.
In the present study, both testosterone and
DHT were more effective in the males than in the
females to induce increases in the gland weight and
the mitotic activity of GCT (Fig. 2 and 5), corres-
ponding to our previous study [11]. In the male
gland, the amounts of androgen receptors (un-
occupied cytosolic receptors plus occupied nuclear
receptors) are approximately 3-fold over those in
the female gland [17]. This may support our
results that the androgen responsiveness of the
mouse gland is sexually different.
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| Byyny, R. L., Orth, D. N. and Cohen, S. (1972)
K. SAWADA AND T.
NOuMURA
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Roberts, M. L. (1974) Testosterone-induced accu-
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Michelakis, A. M., Yoshida, H., Menzie, J., Mura-
kami, K. and Inagami, T. (1974) A radioimmunoas-
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its application to submaxillary gland and kidney
studies. Endocrinology 94: 1101-1105.
Barka, T. (1980) Biologically active polypeptides in
submandibular glands. J. Histochem. Cytochem. 28:
836-859.
Walker, P., Weichsel, M. E. Jr., Hoath, S. B.,
Poland, R. E. and Fisher, D. A. (1981) Effect of
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growth factor (NGF) and epidermal growth factor
(EGF) concentrations in adult female mouse sub-
maxillary gland: dissociation of NGF and EGF
responses. Endocrinology 109: 582-587.
Gresik, E. W., Schenkein, I., van der Noen, H. and
Barka, T. (1981) Hormonal regulation of epidermal
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929.
Kasayama, S., Yoshimura, M. and Oka, T. (1989)
The regulation by thyroid hormones and androgen
of epidermal growth factor synthesis in the subman-
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Gresik, E. W. and MacRae, E. K. (1975) The
postnatal development of the sexually dimorphic
duct system and of amylase activity in the subman-
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422.
Gresik, E. W. (1980) Postnatal developmental
changes in submandibular glands of rats and mice. J.
Histochem. Cytochem. 28: 860-870.
Jayasinghe, N. R., Cope, G. H. and Jacob, S. (1990)
Morphometric studies on the development and sex-
ual dimorphism of the submandibular gland of the
mouse. J. Anat. 172: 115-127.
Sawada, K. and Noumura, T. (1991) Effects of
castration and sex steroids on sexually dimorphic
development of the mouse submandibular gland.
Acta Anat. 140: 97-103.
Sawada, K. and Noumura, T. (1992) Sexually
dimorphic duct system of the submandibular gland
in mouse with testicular feminization mutation
(Tfm/Y). Acta Anat. 143: 241-245.
Takuma, T., Nakamura, T., Hosoi, K. and Kume-
gawa, M. (1977) Binding protein for Sa-
dihydrotestosterone in mouse submandibular gland.
Biochim. Biophys. Acta. 496: 175-181.
Verhoeven, G. (1979) Androgen binding proteins in
15
16
Androgens and Mouse Submandibular Gland
mouse submandibular gland. J. Steroid Biochem.
10: 129-138.
Sato, N., Nemoto, T., Baba, R. and Ota, M. (1985)
Dialysis-induced transformation of mouse subman-
dibular androgen receptor. Biochem. Int. 10: 771-
776.
Minetti, C. A. S. A., Valle, L. B. S., Fava-De-
Moraes, F., Romaldini, J. H. and Oliveira-Filho, R.
M. (1986) Ontogenesis of androgen receptors in the
mouse submandibular gland: correlation with the
developmental profiles of circulating thyroid and
17
18
809
testicular hormones. Acta Endocr. 112: 290-295.
Kyakumoto, S., Kurokawa, R., Ohara-Nemoto, Y.
and Ota, M. (1986) Sex difference in the cytosolic
and nuclear distribution of androgen receptor in
mouse submandibular gland. J. Endocr. 108: 267-
iB.
George, F. W., Johnson, L. and Wilson, J. D.
(1989) The effect of a Sa-reductase inhibitor on
androgen physiology in the immature male rat.
Endocrinology 125: 2434-2438.
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ZOOLOGICAL SCIENCE 9: 811-821 (1992)
© 1992 Zoological Society of Japan
Functional Morphology of Feeding Apparatus of the Cookie-Cutter
Shark, /sistius brasiliensis (Elasmobranchii, Dalatiinae)
SHIGERU SHIRAI’ and KAzuHIRO NAKAYA2
‘Department of Zoology, University Museum, University of Tokyo, 7-3-1,
Hongo, Bunkyo-ku, Tokyo 113, and ? Laboratory of Marine Zoology,
Faculty of Fisheries, Hokkaido University, 3-1-1,
Minato, Hakodate 041, Japan
ABSTRACT— External morphology, skeletons, and muscles related to the peculiar feeding mechanism
of Isistius brasiliensis are described and compared with other squaloid sharks. On the feeding function
of this shark, a few characteristic conditions have been emphasized (e.g., fleshy oral margins, scoop-like
lower jaw with a sharp tooth row, and developed basihyal and throat muscle). Serious morphological
differences are, however, found in the whole of neurocranium, viscerocranium, shoulder girdle, and
related musculature. These characteristic conditions are not suitable for the clutching-cutting feeding
mechanism of typical squaloids but should effectively work on scooping the flesh of preys, making a
strong oral vacuum, and rotating the body itself. These functions explain the cause of a crater-like
wound on fish and cetaceans, and /sistius must apply them not only on large preys but on small fish and
squids.
INTRODUCTION
The cookie-cutter shark, Isistius brasiliensis
(Quoy et Gaimard), is a pelagic species of world-
wide distribution throughout tropical and temper-
ate waters. Among squaloid members, it is rather
small reaching a size of 50cm TL. Recently this
shark attracted many oceanological and fishery
scientists because of its curious feeding habit re-
vealed by Jones [1]. Isistius brasiliensis often feeds
by biting chunks out of teleostean swimmers (e.¢.,
dolphinfish, marlin, tuna, albacore, and wahoo),
cetaceans, seals, and large pelagic sharks including
the megacasmids [1-5]. This small shark gives
these preys a crater-like wound, which has been
thought to be caused by the bacteria, parasites, or
cyclostomes [6]. The purpose of this unusual
feeding habit is ambiguous, because I[sistius’s sto-
mach contents are found largely occupied with
pelagic squids, as large as the body of this shark in
most specimens, small fishes including Vinciguerria
(Photichthyidae), and crustaceans [1, 7, 8]. In its
Accepted April 13, 1992
Received February 14, 1992
feeding mechanism, we do not still understand
enough; Jones [1] referred to only a few character-
istic structures, which will be of help to scoop the
flesh of prey and to induce the strong oral vacuum.
The purpose of the present paper is to describe
anatomical features of the feeding apparatus of
Isistius brasiliensis in detail and to compare with
those of closely related groups. Also, we discuss
these functions on the morphological basis.
MATERIALS AND METHODS
Examination of skeletons and muscles of Jsistius
brasiliensis was based on one male and four
females. Several dalatiine species and other
squaloids were dissected for comparison. Collec-
tion information of these specimens is listed below.
Study materials are deposited in HUMZ (Labora-
tory of Marine Zoology, Faculty of Fisheries,
Hokkaido University) and FSFL (Far Seas Fisher-
ies Research Laboratory, Shimizu, Japan). Defini-
tion of the Dalatiinae follows Compagno [5].
Dalatiinae
TIsistius brasiliensis: one male—HUMZ 119287,
425mm TL; four females—HUMZ 69173, 364
812 S. SHIRAI AND K. NAKAYA
mm TL, HUMZ 86585, 480mm TL, HUMZ
87120, 438 mm TL, HUMZ 89900, 516 mm TL.
Squaliolus aliae: HUMZ 119285, male, 185 mm
TL,
S. laticaudus : two females—HUMZ 74972, 250
mm TL, HUMZ 74974, 245 mm TL.
Euprotomicrus bispinatus: HUMZ 90413, male,
197 mm TL.
Dalatias licha: two males—HUMZ 74603, 390
mm TL, HUMZ 95229, 621 mm TL.
Other squaloids
Deania quadrispinosum: FSFL-EE422, female,
325 mm TL.
Etmopterus lucifer: HUMZ 90230, male, 337
mm TL.
Oxynotus bruniensis: HUMZ 91383, male, 545
mm TL.
Somniosus microcephalus :
female, 1,425 mm TL.
Squalus acanthias : HUMZ 87733, male, 495 mm
TL.
Zameus squamulosus : HUMZ 75872, male, 526
mm TL.
HUMZ 112816,
Figure drawings were made in the camera lucida
image with dissected specimens stained by alcian
blue. Anatomical terminology mainly follows
Daniel [9] and Edgeworth [10].
Abbreviations used in figures are as follows:
alc +—anterior upper labial cartilage
am —adductor mandibulae
ams —adductor m. superficialis
bb —basibranchial
bh —basihyal
btp —basitrabecular process
cb —ceratobranchial
ch —ceratohyal
chd —constrictor hyoideus dorsalis
chy —constrictor hyoideus ventralis
co —coracoid
coa —coraco-arcuales
cob —coraco-branchialis
cod —constrictor dorsalis
coh _—coraco-hyoideus
es —eye stalk
fbc —articular fossa of basihyal with ceratohyal
fopp —foramen for ophthalmicus profundus
gco —genio-coracoideus
gcob —f-slip of genio-coracoideus
gm —gum of upper jaw
gop —groove for orbital process
hb —hypobranchial
hm —hyomandibula
hmf —hyomandibular fossa
hyp —hypaxial body muscle
im —intermandibularis
iow —interorbital wall
jhm —yjoint cartilage of hyomandibula
If —lip fold
Ihc —ligamentum hyomandibulo-hyoideum
lb —levator labialis
llc —lower labial cartilage
Imc —ligamentum mandibulo-hyoideum
Ipc —lip cover
ma —mandibula
mfl mandibular flap
mk —mandibular knob of mandibula
op —orbital process
opd —oral pocket
pap —palatine plate of palatoquadrate
pem —pericardial membrane
ple —posterior upper labial cartilage
potp —postotic process
pmg —posterior mouth groove
pnf —posterior nasal fenestra
pq —palatoquadrate
proc —preoral cleft
prop—preoral pouch
qup —dquadrate plate of palatoquadrate
slc —fleshy sack of posterior upper labial car-
tilage
so —suborbitalis
sos —subotic shelf
II —(foramen for) optic nerve
DESCRIPTION
In this section, external and internal characters
of Isistius brasiliensis, which appear to accommo-
date its feeding mechanism directly or indirectly,
are described and compared with the other mem-
bers of Dalatiinae. The condition of the latter (or
the other squaloid sharks if necessary) is noted in
braces.
Anatomy of Cookie-Cutter Shark 813
Fic. 1.
Mouth structure of squaloids. A) ventral view of Jsistius brasiliensis (the lip cover is deleted in right half); B)
ventral view of same when jaws and lips are protruded showing the exposed gum of upper jaw and the lip sack for
labial cartilages; C) lateral view of same; D) ventral view of Squaliolus aliae (the lip cover is deleted in right half);
E) ventral view of Deania quadrispinosum. Scales are 10 mm.
Externals (Fig. 1)
The mouth of Jsistius (Fig. 1A-C) resembles that
of other dalatiines (Fig. 1D) in having a large
cutaneous lip cover (Ipc) and a thick and naked lip
fold (If) at the mouth corner. The lip cover is a
skin continuing from the upper lip near the jaw
symphysis to the posterior mouth groove (pmg),
concealing a deep oral pocket (opk) and preoral
pouch (prop); non-dalatiine squaloids have no lip
cover, and the preoral pouch is exposed as the
preoral cleft (proc; Fig. 1E). The lip fold is
supported by a joint of the posterior upper and
lower labial cartilages (plc, llc); in non-dalatiine
squaloids (Fig. 1E), the lip fold also conceals
another piece of labial cartilage (alc). The pos-
terior mouth groove of Jsistius is deep and extends
posteriorly rearward to the half of distance from
the corner of mouth to the first gill opening as in
Squaliolus and Euprotomicrus.
In Isistius, the lip fold is stout with the smooth
surface and conceals well-developed labial carti-
lages {lip fold is more or less lamellated, and labial
cartilages are somewhat weak in other dalatiines
(Fig. 1D)}. A gum in the upper jaw (gm) is
considerably thickened, and a fleshy sack for the
upper labial cartilages (slc) is made by the upper
lip {such a bulky gum and the fleshy sack are
absent}.
Skeleton
In the neurocranium of Isistius (Fig. 2A-B), the
nasal capsule is somewhat reduced, but the orbit
and otic capsule are well-expanded, with many
modifications, e.g., an unusual opening of nasal
capsule (pnf) at the subnasal fenestra, unchon-
drified interorbital wall around the foramen op-
ticum (II), no eye stalk (es), and no subotic shelf
(sos). Two conditions related to the jaw suspen-
sion are that: (1) the basitrabecular process (btp) is
814 S. SHIRAI AND K. NAKAYA
(A) hmf
fopp Sos
Fic. 2. Neurocrania of two dalatiines. A-B) lateral (A) and ventral views (B) of Isistius brasiliensis; C-D) lateral (C)
and ventral views (D) of Squaliolus laticaudus. Scales are 10 mm.
expanded laterally to form a shelf below the pos-
terior part of orbit {the basitrabecular process is
only a small bulge supporting the orbital process of
palatoquadrate (Fig. 2C-D)}; and (2) the postotic
process (potp) is developed to form the dorsopos-
terior ridge of a deep hyomandibular fossa (hmf)
|the hyomandibular fossa and postotic process are
not so developed}.
The mandibular arch of Isistius (Fig. 3A-B, 4A-
B) is characterized by fairly reduced upper and
voluminous lower jaws, and it is noticeably dif-
ferent from that of other squaloids in the following
respects: (1) the upper teeth are wholly inside the
lower teeth when mouth is closed, and then they
are not engaged each other {upper and lower teeth
are engaged each other at least in part (Fig. 3D)|;
(2) the palatoquadrate (pq) is composed of two
thin and soft pieces, the anterior one (palatine
plate: pap) is capable to warp dorsally, and the
posterior one (quadrate plate: qup) have a minute
orbital process (op) at its anterior end for the loose
palatoquadrate-neurocranium articulation {the
palatoquadrate is a single and stout cartilage with a
higher orbital process, and the palatoquadrate-
neurocranium articulation is firmer}; (3) the man-
dibula (ma; Meckelian cartilage) is thick and
scoop-like, not curved laterally at the posterior
end {the posterior end of mandibula is somewhat
curved laterally to form a fossa for the adductor
mandibulae (am)|; (4) the mandibula has a flexi-
ble, weakly chondrified plate (mandibular flap:
mfl) at its posteroventral edge {absent}; and (5)
the ligamentum mandibulo-hyoideum (Imc) is very
tough, inserted onto the posterior margin of man-
dibula near the joint between the hyomandibula
and ceratohyal {this ligament is weaker and in-
serted onto the medial surface of the mandibula
below the mandibular knob (mk) somewhat sepa-
rated from the hyomandibula-ceratohyal joint}.
The jaw dentition of Jsistius essentially resem-
bles that of the dalatiines in having the dignathic
heterodonty (Fig. 3); upper teeth are lanceolate
arranging quincuncially and the lower teeth are
with a large plate-like crown forming an interlock-
ing tooth blade. In Jsistius, the lower tooth rows
almost extend to the point of jaw joint (articula-
tion of upper and lower jaws) to form a semicircu-
lar saw as a fringe of the scoop-like lower jaw {the
most posterior lower tooth is moderately separate
from the jaw joint{. The lower teeth are enor-
mously large and have a triangular, fully erected
cusp without a regular serration {lower teeth have
a more or less inclined cusp with or without
serration|. In contrast, upper teeth are weak, and
Anatomy of Cookie-Cutter Shark 815
(}
\
1
1
|
i
\
Fic. 3. A) Neurocranium and mandibular and hyoid arches of Isistius brasiliensis showing jaw suspension (the
hyomandibula is drawn from dorsal view), and lateral view of hyoid arch as inset (right upper); B) dorsal view of
mandibular arch of Jsistius brasiliensis; C) same as (A) in Squaliolus laticaudus; D) same as (B) in Squaliolus
laticaudus. Scales are 10 mm.
816 S. SHIRAI AND K. NAKAYA
the functional tooth rows are not supported by the
palatoquadrate in part |upper tooth rows are
entirely supported by the palatoquadrate}.
The hyoid arch (Figs. 3A, 5A) of Jsistius is also
modified considerably. The hyomandibula (hm),
like Dalatias, is short, flat, and directed laterally,
not inclined posteriorly {the hyomandibula is
somewhat longer, compressed, and directed pos-
tero-latero-ventrally (Fig. 3C)|. The proximal ter-
minus of hyomandibula is bifurcate, and its pos-
teroventral condyle is composed of a separate
cartilaginous piece (jhm) {it is also slightly bifur-
cate, but without a separate cartilage}. The distal
terminus of hyomandibula has a small expansion to
receive the mandibular knob of mandibula (mk) at
its anterior edge and a simple depression for the
articulation with the ceratohyal at its posterior
edge. Thus, the hyomandibula-ceratohyal arti-
culation is somewhat behind the mandibula-
hyomandibula articulation {this feature is also
seen in Dalatias, but not so separate as in Isistius ;
the distal terminus of the hyomandibula is distinct-
ly bifurcate for the articulation with the ceratohy-
al, and the hyomandibula-ceratohyal articulation is
very close to the mandibular knob}. The ceratohy-
nem
————
Fic. 4. Head musculature of two dalatiines.
al (ch) of Jsistius is very stout, connected with the
hyomandibula by a thick ligament (ligamentum
hyomandibulo-hyoideum: lhc); this ligament arises
from the basicranium below the hyomandibular
fossa, covering the posterodorsal surface of the
hyomandibula, and it is inserted on the anterodor-
sal surface of ceratohyal {the ceratohyal is also
developed, but such a ligament is absent except
Dalatias|. The large quadrangular basihyal (bh)
has a simple concavity (fbc) at its posteroventral
surface to receive the anterior terminus of the
ceratohyal (Fig. 5A) |the basihyal is smaller,
generally trapezoid in shape, and the fossa for
articulation with the ceratohyal is situated on its
posterolateral margin (Fig. SB-C)}.
Isistius has strongly reduced branchial arches,
and the basibranchial copula (hypobranchials plus
basibranchials) is almot absent (Fig. SA). The first
ceratobranchial (cb1) is not articulated with the
basihyal, situated far posterior from it |in Dalatias,
the ceratobranchial-basihyal articulation is also
absent, but the first ceratobranchial is supported
by the hypobranchial as a transverse bar (hb1: Fig.
5C); in other squaloids, the first ceratobranchial is
connected with the basihyal tightly or loosely (Fig.
AX
Mi
\
\Y
wit
A-B) lateral (A) and ventral views (B) of J/sistius brasiliensis; C-D)
lateral (C) and ventral views (D) of Squaliolus laticaudus (B and D: mandibula is removed in upper half, above
double solid line). Scales are 10 mm.
Anatomy of Cookie-Cutter Shark 817
5B)}. Jsistius is unique in having an elongate
separate hypobranchial in the fifth arch (hb5), to
which the large, paddle-shaped ceratobranchial
(cb5) is loosely connected |the fifth arch does not
have a discrete hypobranchial}. The gill raker is
completely absent {present in most other
squaloids} .
The shoulder girdle (Fig. 4B) of Jsistius is char-
acterized by the fragile and curiously curved cora-
coid (co). It courses medially for a short distance,
turns anteriorly at a right angle, and extends
anteroventrally to form a peculiar hairpin turn at
the symphysis that forms the pericardial floor |the
coracoid bar is a stout transverse bar, and this is
not strongly curved (Fig. 4D)}.
Musculature
The mandibular muscles (Fig. 4A-B) are also
modified in Jsistius as follows: (1) the adductor
mandibulae (am) is very reduced, and its insertion
onto mandibula is restricted slightly below the jaw
joint via tough tendinous tissue {the adductor
mandibulae is housed at the whole posterior half of
mandible (Fig. 4C)}; (2) the thick suborbitalis (so)
is inserted onto the tendinous tissue of the adduc-
tor mandibulae {the suborbitalis is inserted onto
the anterior part of mandibula with (Dalatias) or
Fic. 5.
Basal portion of hyoid and branchial arches with the coraco-branchiales of three dalatiines.
brasiliensis (left—ventral view showing the coraco-branchialis 1; right—ventral view showing the coraco-
branchialis 5; B) Squaliolus laticaudus (ventral view); C) Dalatias licha (ventral view). Scales are 10 mm.
without (Euprotomicrus and Squaliolus) a tendon
for insertion (Fig. 4C-D)}; (3) the adductor m.
superficialis (ams) is completely separate from the
adductor mandibulae, arising from the outer sur-
face of the mandibula {this muscle is usually a
subdivision of the adductor mandibulae, situated
on the otic process of palatoquadrate}; (4) a
unique thin muscle, the levator labialis (IIb), arises
from the otic process of palatoquadrate and is
inserted on the medial edge of the posterior mouth
groove {absent}; and (5) the anterior and pos-
terior extremities of the constrictor hyoideus dor-
salis (chd) are inserted on the mandibula | the ante-
rior edge of this muscle is inserted onto the pala-
toquadrate, not reaching the mandibula}.
The hypobranchial longitudinal muscles, occu-
pying the space below the oral cavity and pharynx,
are noticeably modified in Jsistius. The genio-
coracoideus (gco) is thin and broad, tapering for-
ward, and originates from the ventral surface of
the hypaxial muscle below the origin of pectoral fin
(Fig. 4B). Another slip of muscle (gcob), arising
from the anterior curved surface of the coracoid
and inserted onto the mandibula with the genio-
coracoideus, is present (genio-coracoideus 2) {the
genio-coracoideus is an unpaired, narrow muscle
bundle that arises from coracoid symphysis or
A) Isistius
818 S. SHIRAI AND K. NAKAYA
pericardial membrane, and the genio-coracoideus
B is absent (Fig. 4D)}. The rectus cervicis, often
subdivided into the anterior (=coraco-hyoideus:
coh) and posterior halves (=coraco-arcuales:
coa), is expanded well and is directly connected
with the hypaxial muscle posteriorly {the rectus
cervicis arises from the coracoid, and is not directly
connected with the hypaxial muscle}. The coraco-
branchiales (cob) are simplified, composed of two
slips (Fig. 5A); one (cobl) is a wide and flat,
arising from the pericardial membrane and in-
serted on the basihyal; the other (cob5) originates
on the dorsal side of the fascia of coraco-arcuales,
and its insertion is on the fifth hypobranchial and
ceratobranchial {the coraco-branchiales are com-
posed of five slips (Fig. 5B); their origins are the
fascia of rectus cervicis (first) and the pericardial
membrane or the anterior surface of coracoid
(others), and insertions are, from front, the
basihyal, the ceratobranchial and hypobranchial of
the second, third, fourth, and fifth arches; Dalatias
(Fig. 5C) also has two separate coraco-branchiales
like Jsistius, but the posterior slip arises from the
coracoid}.
DISCUSSION
We can often find crater wounds on the surface
of tunas and swordfishes in a fish market (Fig. 6).
It is round or oval with the size of 5 cm by 7 cm at
the most. Jones [1] discussed the probability of the
relation between this wound and the feeding
mechanism of Jsistius. His arguments on the
feeding function of this shark are summarized as
follows:
(1) Isistius gives such a wound by pushing its
lower tooth row against the body of prey and then
scooping the flesh. In his experiment with a dead
fish, the scoop-like lower teeth of Jsistius can
produce a crescentic wound, which is very similar
in size and shape to that often observed in tunas
caused by the incomplete attack.
(2) Isistius can make a strong oral vacuum when
the tongue (basihyal) is pulled back by the volumi-
nous throat muscle (rectus cervicis). Concurrent-
ly, the shark closes its spiracles and protrudes lips
completely around the mouth.
(3) The body rotation should be facilitated by the
Fic. 6. Crater wounds on sides of tunas and swordfish
at the Kesennuma Fish Market, Miyagi prefecture,
Japan (A-B, Tunnus alalunga; C, Xiphias gladius).
A wound in (C) penetrates into the body cavity.
Each wound is 5 to 7 cm in a major axis. (B and C:
photo by Toshiro Chiba).
drag of water when J/sistius heads on the prey as
expected in many examples. At the next moment,
the body of Jsistius would rotate around the point
of attachment until the shark is oriented in the
same direction as the prey. The crater wound is
then completed.
Morphological features of this shark can explain
these functions as mentioned below.
On the first hypothesis, Jones [1] noticed the
peculiarity of dentition in Jsistius. This shark
shares a typical dignathic heterodonty with many
other squaloids, which should work for the clutch-
ing-cutting-type feeding habit [11]. Squaloid
sharks (Figs. 3C, 4C) generally hold food items by
the teeth of both jaws, and cut them by sharp
lower teeth. Developed adductor mandibulae
plays the main role in this function. In Jsistius
(Figs. 3A-B, 4A-B), upper and lower teeth cannot
be engaged each other. Upper and lower jaws are
ill-balanced in size, and the palatoquadrate is
Anatomy of Cookie-Cutter Shark 819
subdivided into the anterior and posterior halves.
The adductor mandibulae is small, not widely
inserted on the lateral surface of lower jaw. At the
collision with a prey, the lower jaw is given a
strong shock (reaction of the collision), but it must
keep the appropriate angle to penetrate the flesh
with the lower teeth. The massive suborbital
muscle, with the reduced adductor mandibulae,
appears to support this function.
In contrast with the tough lower teeth, the role
of upper teeth seems rather moderate. Jones [1]
observed several scratches produced by Isistius’s
upper teeth. These are opposite to a crescentic
wound caused by lower teeth, at barely inside the
crater if this attack were complete. The flexible
upper jaw might be bent at the middle portion to
make the oral area greater at the collision. Upper
teeth are thus effectively anchored on the prey to
prevent the shark body whisked off during the
gouging.
Such unique jaw morphology is apparently not
suited to clutch and masticate foods. The scoop-
like tooth row of the lower jaw along the ventral
surface of body thus appears more appropriately
designed to gouge out the flesh of prey as sug-
gested by Jones [1]. Jsistius usually feeds small
pelagic fish and squids [1, 7, 8], and this scooping
function also should occur for these small preys.
Second, Jones [1] explained that the strong oral
vacuum is induced by the function of a large
basihyal, the developed rectus cervicis, spiracles,
and lips. Thickened labial fold and gum in the
upper jaw and the flexible upper jaw must be
useful to fit the oral margin to the shape of food.
We found the unusually curved shoulder girdle,
and this allows the rectus cervicis directly contacts
with the thick hypaxial muscle. The oral vacuum
will be thus induced by the contraction of whole
ventral muscle of body. We also noticed that the
basal portion of branchial arches is incomparably
reduced, especially in the separation between the
first ceratobranchial and basihyal. This must be
concerned with stronger and smoother retraction
of the basihyal.
Isisttus has another mechanism to make the
stronger oral vacuum with ingenuity (Fig. 7). In
most shark groups, the lower jaw is lowered main-
ly by the function of the whole hyoid arch and
muscles inserted onto the basihyal (rectus cervicis
and coraco-branchialis 1). As shown in Figure 7C,
Fic. 7.
Diagrams of the retraction of basihyal and movements of the associated parts of (A-B) Jsistius and (C-D)
Squaliolus (upper—lateral view; lower—dorsal view). Black dots show the following points: /, the ligamentous
connection between ceratohyal and mandibula; 2, the articulation between mandibular knob and hyomandibula;
3, the hyomandibula-ceratohyal articulation; and 4, the basihyal-ceratohyal articulation.
820 S. SHIRAI AND K. NAKAYA
the ligamentum mandibulo-hyoideum is usually
short and fastens the ceratohyal and mandibular
rigidly (point /) somewhat below the hyomandibu-
la-ceratohyal articulation (point 3). The point 3 is
the center of rotation of the ceratohyal, and it is
very near the center of rotation of the mandibula
(point 2). When the basihyal is retracted (Fig.
7D), therefore, the mandibula will be lowered at
the almost same degree as the rotation of ceratohy-
al, and the oral cavity does not expand so strongly.
In Isistius (Fig. 7A), the point 3 is characteristical-
ly posterior to the point 2 by the width of hyoman-
dibula. The ligamentum mandibulo-hyoideum (/)
joins the posterior margin of mandibula with the
ceratohyal near the hyomandibula-ceratohyal ar-
ticulation (3). The anterior part of ceratohyal thus
can be somewhat free from the mandibula.
Moreover, the basihyal has a semi-globate fossa
for the articulation with ceratohyal (point 4), and
it gives the flexibility to the basihyal-ceratohyal
When the basihayl is pulled back
(Fig. 7B), the anterior end of the ceratohyal can
swing posterointernally like man’s wrist when
elbows are spread out. Therefore, the basihyal can
move backward more strongly than the lowering of
mandibula, and the oral cavity is expected to be
more enlarged than the other squaloids.
The bigger the oral cavity is, the stronger the
oral vacuum should be induced. The shape and
size of mandibula and basihyal clearly show that
Isistius essentially has a larger oral cavity. The
movement of ceratohyal mentioned above will
serve to expand it further. In addition, the mod-
ification of the hyomandibula should be also re-
lated with this function. This cartilage is directed
posterolaterally when the mouth is closed and has
a separate cartilaginous piece at the proximal end.
The hyomandibular fossa on the otic capsule is
well developed. These structures probably make
the hyomandibula lower more strongly to enlarge
the oral cavity.
On the third hypothesis (mechanism of body
rotation), Jones [1] explained that /sistius utilizes
the locomotion of prey and the drag of water for
gouging. It is reasonable for the fast swimmers.
However, I/sistius attacks the slower fishes and
cetaceans, and even the dead fishes. Almost all the
wounds on tunas and swordfishes we observed
articulation.
were so fresh with bleeding that these wounds
would have been given after they were restrained
by the fishing gear (long line) or dead on hooks
(Fig. 6). The body rotation of Jsistius should be
thus more spontaneous than Jones considered.
The body itself must be twisted by the differential
contraction of right and left body muscles.
We consider that these three basic functions for
feeding occurs in a very moment. This small shark
stabs the prey with the sharp lower teeth at the
collision. Immediately after this, the ventral body
muscle must be suddenly contracted to pull the
basihyal back and the oral vacuum is induced in
order to stick the mouth to the prey. The contrac-
tion of body muscle should simultaneously create a
twisting moment of the body itself with or without
the help of the drag of water. A half turn of body
probably makes a complete crater-like wound.
Two dwarf dalatiine shark genera, Euprotomi-
crus and Squaliolus, have the similar habitat and
outlook to Jsistius, and these were often compared
with each other [8, 12, 13]. The feeding mecha-
nisms of Euprotomicrus and Squaliolus, however,
should be the typical squaloid-type (clutching-
cutting type) [11], because their feeding apparatus
is essentially same as that of squaloids. Isistius
rather share some characteristic conditions in the
feeding apparatus with another dalatiine shark,
Dalatias licha, i.e., the hyomandibula-ceratohyal
articulation somewhat behind the mandibular
knob, the first ceratobranchial widely apart from
the basihyal, and reduced coraco-branchiales com-
posed of two muscle slips. However, Dalatias’s
palato-quadrate, arrangement of upper and lower
tooth rows, basihyal, and related musculature are
well similar to those of other squaloids, and it is
unlikely that it takes same feeding manner as
Isistius does. Two other rare dalatiine sharks,
Euprotomicroides and Heteroscymnoides, whose
feeding apparatus is hardly known, cannot scoop
out the flesh of fish either because of their typical
dalatiine dentition [5, 14]. A culprit of the crater
wound is thus clearly identified, the cookie-cutter
shark.
ACKNOWLEDGMENTS
We would like to thank Kunio Amaoka (Laboratory of
Anatomy of Cookie-Cutter Shark 821
Marine Zoology, Faculty of Fisheries, Hokkaido Uni-
versity) who gave us considerable support and sugges-
tions during this study. We are also indebted to Stuart G.
Poss (Gulf Coast Research Laboratory Museum, Ocean
Springs) for constructive reading of the manuscript.
Toshiro Chiba (Kesennuma City) kindly took photo-
graphs of crater wounds. Keiichi Matsuura, Richard
Rosenblatt, “H. J.” Walker, Jr., Yasunori Sakurai,
Shigeyuki Kawahara, and Harumi Yamada provided
specimens for comparative studies.
REFERENCES
1 Jones, E. C. (1971) Isistius brasiliensis, a squaloid
shark, the probable of crater wounds on fishes and
cetaceans. Fish. Bull., 69: 791-798.
2 Van Utrecht, W. L. (1959) Wounds and scars on the
skin of the common porpoise, Phocaena phocaena
(L.). Mammalia, 23: 100-122, pls. 6-9.
3 Baker, A. N. (1985) Pygmy right whale, Coperea
marginata (Gray, 1846). In “Handbook of marine
mammals, vol. 3. The sirenians and baleen whales”.
Ed. by S. H. Ridgeway and R. Harrison, Academic
Press, London, pp. 345-354.
4 Le Boeuf, B. J., McCosker, J. E., and Hewitt, J.
(1987) Crater wounds on northern elephant seals:
the cookiecutter shark strikes again. Fish. Bull.,
U.S., 85: 387-392.
5 Compagno, L. J. V. (1984) FAO species catalogue.
Vol.4. Sharks of the world. An annotated and
illustrated catalogue of shark species known to date.
Part 1. Hexanchiformes to Lamniformes. FAO Fish.
Synop. (125) 4: 1-249.
6 Pike, G. C. (1951) Lamprey marks on whales. J.
13
14
Fish. Res. Board Canada, 8: 275-280.
Strusburg, D. W. (1963) The diet and dentition of
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ment in other sharks. Copeia, 1963: 33-40.
Hubbs, C. L., Iwai, T. and Matsubara, K. (1967)
External and internal characters, horizontal and
vertical distribution, luminescence, and food of the
dwarf pelagic shark, Euprotomicrus bispinatus. Bull.
Scripps Inst. Oceanogr., 10: i-vi+ 1-64, pls. 1-8.
Daniel, J. F. (1934) The elasmobranch fishes. 3rd
ed. Univ. California Press., Berkeley, California, xii
+332 pp.
Edgeworth, F. H. (1935) The cranial muscles of
vertebrates. Cambridge Univ. Press, Cambridge,
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Cappetta, H. (1987) Handbook of Paleoichthyolo-
gy. Volume 3B. Chondrichthyes II. Mesozoic and
Cenozoic Elasmobranchii. Gustav Fischer Verlag,
Stuttgart, i11+193 pp.
Hubbs, C. L. and McHugh, J. L. (1951) Rela-
tionships of the pelagic shark Euprotomicrus bispi-
natus, with description of a specimen from off
California. Proc. Calif. Acad. Sci., 27: 159-176.
Seigel, J. A. (1978) Revision of the dalatiid shark
genus Squaliolus: anatomy, systematics, ecology.
Copeia, 1978: 602-614.
Stehmann, M. and Krefft, G. (1988) Results of the
research cruises of FRV “Walther Herwig” to South
America. LXVIII. Complementary redescription of
the dalatiine shark Euprotomicroides zantedeschia
Hulley and Penrith, 1966 (Chondrichthyes, Squali-
dae), based on a second record from the Western
South Atlantic. Arch. FischWiss., 39: 1-30.
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ZOOLOGICAL SCIENCE 9: 823-834 (1992)
Neuropeptide Y Innervation of Cerebral Arteries
in Microchiropteran Bats
Korcut ANpbo! and Suser ARAI*
‘Biological Laboratory, Liberal Arts, Kyushu Sangyo Universtiy, Matsukadai,
Higashi-ku, Fukuoka, 813, and *Department of Oral Anatomy, Kyushu
Dental College, Manazuru, Kokura-Kita, Kitakyushu, 803, Japan
ABSTRACT— The distribution and origin of neuropeptide Y (NPY) immunoreactive nerves in the walls
of the cerebral arteries were investigated in two microchiropteran species. The majority of cerebral
perivascular NPY nerves originated in the sympathetic internal carotid nerve emanating from the
superior cervical ganglion. The NPY nerves ran in the carotid canal, intermingled with parasympathetic
axons containing vasoactive intestinal polypeptide (VIP), and reached the circle of Willis along the
internal carotid artery. In addition, a part of cerebrovascular NPY nerves entered the cranial cavity
along the internal ethmoidal and vertebral arteries. The presence of NPY nerves with VIP but not with
tyrosine hydroxylase, a key enzyme for noradrenaline synthesis, seems to indicate that some of the
cerebral perivascular NPY nerves are not of sympathetic in nature. The supply of NPY nerves to the
cerebral arterial tree of the small bat was the richest among mammalian species studies up to date.
Compared with the general mammalian pattern, NPY nerve supply was much more prominent in the
vertebrobasilar system, particularly along the walls of the posterior cerebral and basilar arteries, than in
© 1992 Zoological Society of Japan
the internal carotid system.
INTRODUCTION
Many histochemical studies have demonstrated
dual innervation of cerebrovascular bed by sym-
pathetic noradrenergic and parasympathetic cho-
linergic nerves in a variety of mammalian species
[1-4]. Besides these two classical populations of
cerebrovascular nerves, recent advance in im-
munohistochemical techniques revealed that cere-
bral vessels are innervated by populations of
neurons containing various biogenic active pep-
tides, such as neuropeptide Y (NPY), vasoactive
intestinal polypeptide (VIP), substance P (SP),
and calcitonine gene-related peptide (CGRP) [5,
6]. Of these neuropeptide-containing nerves, NPY
nerves have been identified to be largely sym-
pathetic in nature (coexistence of NPY with norad-
renaline (NA) in the same cerebral perivascular
nerves) and mainly originate from the superior
cervical ganglion (SCG) [7-10]. Pharmacological-
ly, NPY is known to exert a potent vasoconstrictor
Accepted January 6, 1992
Received June 4, 1992
effect on cerebral arteries, with a long lasting
action that is resistant to a-adrenoceptor and cho-
linergic receptor blockages [11-13]. NPY may also
potentiates prejunctionally the vasoconstrictor
effect of noradrenergic nerves [10, 12]. Thus, it
has now been emphasized that NPY, as well as
other neuropeptides (VIP and SP for vasodilator,
and CGRP for vasoconstrictor), participates in the
regulation of the cerebral circulation as a neuro-
transmitter or neuromodulator [14-16]. The
above-mentioned morphological and pharmacolo-
gical evidence concerning the cerebrovasular pep-
tidergic innervation is issued mostly from labora-
tory mammals, especially from the rat, cat and
guinea pig.
As a general angioarchitecture for mammalian
cerebral circulation, the internal carotid and ver-
tebral arteries (ICA, VA) enter the cranial cavity
through the carotid and vertebral canals, respec-
tively, and send many branches to the brain. The
branches arising from the ICA are distributed to
most parts of cerebrum and diencephalon, forming
the anterior part of cerebral arterial tree, the
internal carotid system (ICS). The right and left
824 K. ANDO AND S. Aral
VA joint together to construct the basilar artery
(BA) on the midline of medulla oblongata. The
branches from the VA and BA supply the brain
area caudal to the caudal part of cerebrum, form-
ing the posterior part of the arterial tree, the
vertebrobasilar system (VBS). Rostrally, the right
and left ICS are jointed to each other in front of
the optic chiasm by the anterior communicating
artery. Caudally, the right and left terminal bran-
ches of the BA are jointed ipsilaterally to the
intracranial part of the internal carotid artery, the
cerebral carotid artery (CCA), by the posterior
communicating artery, or by the posterior ramus.
Thus, the ICS and VBS connect with each other at
the base of brain to build up the circle of Willis.
In microchiropteran species, the cerebral vascu-
lar system shows structural difference from the
basic mammalian pattern [17-20]: the major cere-
bral arteries of the ICS are so fine as to be almost
non-functional, while the arteries of the VBS are
markedly well-developed. Consequently, blood
supply to the brain is predominantly supplied by
the VBS. Furthermore, the heart rate and body
temperature of small bats strikingly increase or
decrease in flying and hibernating periods [19].
Such special behaviour and ecology of these mam-
mals should cause the seasonal change of physiolo-
gical conditions in their brains, and may also
involve cerebral blood flow that is unusual in other
mammals. Therefore, it is a matter of interest to
explore the neurogenic mechanisms by which the
bat cerebral vascular bed is regulated, and to
ACA anterior cerebral artery
ACA1 rostral part of the anterior cerebral artery
ACA2 caudal part of the anterior cerebral artery
AR anterior ramus
BA basilar artery
C cochlea
CA common carotid artery
CC carotid canal
ECA external carotid artery
CCA cerebral carotid artery
IAA internal acoustic artery
ICA internal carotid artery
ICS internal carotid system
internal ethmoidal artery
middle cerebral artery
OC optic chiasm
posterior cerebral artery
proximal part of the posterior cerebral artery
compare them with those of other mammals. Such
a comparative study provides new insights that
allow further comprehension of the neuronal in-
fluence on the mammalian cerebral circulation.
The aim of the present study was to investigate the
distribution, origin and pathway of NPY nerves
surrounding the cerebral arteries of the bent-
winged bat and the greater horseshoe bat, and to
correlate them with those of noradrenergic and
VIP nerves using double immunostaining.
MATERIALS AND METHODS
Tissue preparation
Four greater horseshoe bats (Rhinolophus fer-
rumequinum nippon) and eight bent-winged bats
(Miniopterus schreibersii fuliginosus) were used in
this study. The indivuduals examined were of
either sex; all were adults. The animals were
anesthetized with ethyl ether and perfused through
the left ventricle of the heart with ice-cold Ringer’s
solution, followed by 50 ml of Zamboni’s fixative
[21]. The major cerebral arteries, the internal
carotid and vertebral arteries outside the cranial
cavity, and the superior cervical ganglia were
rapidly dissected out, and postfixed in the same
fixative for 18h at 4°C. They were washed with
70% ethanol, dehydrated in a graded ethanol
concentration, and placed in cold 0.1 M phosphate
buffer (PB, pH 7.4). The specimens were then
immersed in PB containing 10% and 20% sucrose
PCA2 distal part of the posterior cerebral artery
PR posterior ramus
S stapes
SA stapedial: artery
SCG superior cervical ganglion
SCA superior cerebellar artery
SICN sympathetic internal carotid nerve
TB terminal branch of the basilar artery
VA vertebral artery
VAI rostral part of the vertebral artery
VA2 caudal part of the vertebral artery
VBS vertebrobasilar system
I rhiencephalon
II cerebrum
Ill cerebellum
IV medulla oblongata
Vv diencephalon
Cerebrovascular NPY Innervation in Bats 825
for 2 days each at 4°C. For whole-mount prepara-
tion, the cerebral arteries, and the extracranial
internal carotid and vertebral arteries were put in
0.1M phosphate-buffered saline (PBS, pH7.2),
and stripped of pia mater or outer adventitial
connective tissue. The nerve bundles accompany-
ing the two extracranial arteries were also dis-
sected and carefully stripped of their perineural
connective tissue. They were then stored in PBS
for 1-24h at 4°C. For sectioning, the superior
cervical ganglion and small blocks of the brain
parenchyma containing intracerebral vessels were
soaked with 10% gelatine PB for 1-2 h in vacuo at
38—40°C after the treatment with sucrose PB. The
gelatine-embedded samples were quickly frozen in
isopentane or acetone chilled with dry ice, sec-
tioned at 20 um thickness in a cryostat, and stored
in PBS at 4°C.
Immunohistochemical protocol
Whole-mount preparations and free-floating
sections were processed for immunohistochemistry
by avidin-biotin immunoperoxidase [22] or indirect
immunofiuorescence [23] techniques. Before the
antigen-antibody reaction of peroxidase immuno-
histochemistry, the specimens were treated for 1 h
at room temperature with PB containing 0.1%
hydrogen peroxide to inhibit endogenous perox-
idase in tissues, and washed with PBS containing
0.3% Triton X-100 (PBST). The specimens were
incubated for three days at 4°C in polyclonal rabbit
NPY antiserum (Code no. 06246; Cambridge Re-
search Biochemicals Lts., Harston, England) at a
dilution of 1:500, followed by washing in cold
PBST. For peroxidase immunohistochemistry, the
specimens were incubated with biotinylated anti-
rabbit immunoglobulin G (IgG) and then with
rabbit avidin-biotin-peroxidase complex (Vectas-
tain kit, Vector Lab., England) for 1 h each time at
room temperature. They were stained with 3,3’-
diaminobenzidine for 5-10 min [24], mounted on
glass slides coated with chrome-alum gelatin, and
examined under a light microscope. For im-
munofluorescence staining, the whole-mounts and
sections treated with the NPY antiserum were
incubated for 1-2h at 37°C in sheep anti-rabbit
IgG conjugated with fluorescein isothiocyanate
(FITC) (Cappel, PA) at a dilution of 1:100. They
were then washed with PBS, mounted in glycerine-
PB mixture (1:1), and examined in a fluorescence
microscope.
To identify the location of NPY and two other
neuronal substances (NA, VIP) in the same speci-
men, double-immunostaining was carried out with
primary antisera raised in different species and
second antisera labelled with different
fluorochromes. Tissues were first incubated with
rabbit NPY antiserum and then with sheep anti-
rabbit IgG conjugated with FITC. Next, they were
exposed to monoclonal mouse antiserum against
tyrosine hydroxylase (TH), a key enzyme for NA
synthesis, (Code no. 10968620-01; Boehringer
Mannheim, W. Germany) at a dilution of 1:1 (40
vg/ml), or polyclonal guinea pig antiserum against
VIP (Code no. M-8701-1, Milab, Malmo, Sweden)
at a dilution of 1:1250, and then to rabbit anti-
mouse (Amersham, England) or goat anti-guinea
pig IgG (E-Y Laboratories, INC, San Mateo, CA)
conjugated with Texas Red (TR) at a dilution of
1:200. Tissues were mounted in the PB-glycerine,
and viewed under a fluorescence microscope.
Coexistence of NPY and TH or VIP was estab-
lished by switching between the filter combinations
for FITC-and TR-induced fluorescence.
To check the specificity of NPY, TH or VIP
immunoreactivity, the following control experi-
ments were performed: (1) incubation with the
serum from non-immunized rabbit, mouse or
guinea pig as primary antiserum; (2) incubation
omitting the primary antiserum in the first step of
immunohistochemical procedures; (3) incubation
with primary antisera preabsorbed with the corres-
poinding peptide (more than 10 g/ml diluted
antiserum) for 24 h at 4°C. When the tissues were
treated in one of the three ways, no specific
immunoreactivity was found in neurons.
RESULTS
Course of the nerve bundles accompanying the
internal carotid artery within the tympanic cavity
In the greater horseshoe bat and the bent-
winged bat, the ICA and the sympathetic nerve
bundle accompanying it (the sympathetic internal
carotid nerve (SICN) emanating from the SCG)
826 K. ANDO AND S. ARAI
(Rostral side)
(Caudal side)
1
Fic. 1. Course of the internal carotid artery, sympathe-
tic internal carotid nerve, and microganglion-
containing nerves in the typmanic cavity of the
bent-wingted bat. Asterisks indicate microganglia in
the nerve bundle accompanying the sympathetic
internal carotid nerve.
2A eu air 2B
entered the tympanic cavity immediately after
their branching. They then ran rostro-medially on
the surface of the cochlea in close association with
each other, and reached the cranial cavity through
the carotid canal (Fig. 1). In the bent-winged bat,
another delicate nerve bundle, with two to three
microganglia, accompanied the tympanic part of
the SICN at the level where the ICA on the
cochlea gave off a branch to the stapes, and then
ran towards the carotid canal.
Arterial Supply of the brain
The cerebral arterial systems of the greater
horseshoe and bent-winged bats were basically
identical to each other. The middle and anterior
cerebral arteries (MCA, ACA), and the internal
ethmoidal artery (IEA) belonging to the ICS were
distributed only to the ventral surface of cerebrum
(Fig. 2A). Compared to this, the posterior cere-
bral artery (PCA) arising from the VBS ran caudo-
rostrally within the longitudinal cerebral fissure to
reach as far as the rhiencephalon, and during its
course sent numerous branches to most parts of
the medial, dorsal, and lateral surfaces and paren-
chyma of cerebrum (Figs. 2BC).
2c
Fics. 2A, B, C. The arterial supply to the brain of the greater horseshoe bat, from ventral (A), dorsal (B), and
midsaggital (C) aspects.
Cerebrovascular NPY Innervation in Bats 827
Immunohistochemistry
In the two microchiropteran species studied,
most of cell bodies in the SCG was immunoreac-
tive for NPY (Fig. 3A), and no VIP immunoreac-
tivity was found in this sympathetic ganglion (Fig.
3B). The tympanic and carotid parts of the ICA
had a scarce supply of NPY nerves, and the SICN
accompanying these cranial parts of the ICA ex-
pressed intense NPY immunoreactivity over its
entire length (Figs. 4, 5). In the bent-winged bat,
two to three microganglia in the nerve bundle
accompanying the tympanic part of the SICN
comprised a large number of cell bodies with VIP
Fics. 3A, B. Fluorescence photomicrographs of cross-sections of the superior cervical ganglion of the greater
horseshoe bat. Double immuno-fluorescent staining for NPY (A) and VIP (B). 150.
Fics. 4, 5.
Photomicrographs of the internal carotid artery of the bent-winged bat showing NPY immunoreactivity in
the sympathetic internal carotid nerve (arrows). Tympanic (4) and carotid (5) regions. X67.
Fics. 6,7. Fluorescence photomicrographs of whole-mounts showing VIP (6) and NPY (7) immunoreactivity in the
microganglia (asterisks) in the nerve bundle accompanying the sympathetic internal carotic nerve at the rostral
part of the typmpanic cavity of the bent-winged bat. Fig. 6x 100; Fig. 7<200.
828 K. ANDO AND S. Aral
Cerebrovascular NPY Innervation in Bats 829
(40-S0 cells per ganglion) as reported previously
[25], but had no cell bodies with detectable level of
NPY (Figs. 6, 7). Such microganglia were not
detected in any parts of the tympanic cavity of the
greater horseshoe bat.
No appreciable difference in the density of NPY
nerves was found between the corresponding ma-
jor cerebral arteries of two bats. As was the case in
the carotid part of the ICA, the CCA had a very
poor supply of NPY nerves. However, several
thick fibre bundles with NPY, which also exhibited
VIP immunoreactivity, reached the circle of Willis
through the CCA (Fig. 8), and provided abundant
NPY fibres to the major arteries of both the ICS
and VBS. NPY axons from these fibre bundles
mainly extended in a caudal direction along the
posterior ramus, so that the supply of NPY nerves
was much more prominent in the major arteries of
the VBS as compared to those of the ICS (Table 1,
Figs. 8-13). The immunoreactive nerves were
particularly rich along the walls of the PCA to BA,
formed complicated meshworks which were orga-
nized mainly from thin varicose fibres (Figs. 11,
12). The distal PCA, the major branches of the
BA, such as the superior cerebellar and internal
acoustic arteries, and the rostral part of the VA
were also furnished with well developed networks
of NPY nerves (Figs. 11-13). The VA just after
entering the cranial cavity was pooly supplied with
NPY nerve fibres, but one or two fibre bundles
with NPY were present on its wall, and ascended
TABLE 1.
bent-winged bat
towards the BA (Fig. 14). As to the ICS, the walls
of the anterior ramus to the caudal part of the
ACA were invested with a rich or moderate num-
ber of NPY nerves (Fig. 8A), but the immunoreac-
tive nerves became distinctly sparse towards the
rostral part of the ACA, the MCA, and the IEA
(Figs. 9, 10). In addition, one or two thin fibre
bundles showing weak NPY immunoreactivity
were seen to run along the IEA (Fig. 10), and their
fibre branches could be traced to the confluence of
the ACA and MCA (Fig. 10). There were no
ganglionic structures with NPY along the walls of
the whole lengths of the extracranial ICA and VA,
and of the major cerebral arteries at all parts of the
small bat brain (see Fig. 2).
Nearly all of the small branches from the major
cerebral arteries of the VBS were supplied with
NPY nerves (Fig. 15). Similarly, NPY fibres
penetrating into the brain parenchyma via the pial
arteries could often be observed in the walls of
intracerebral arteries and arterioles of this arterial
system, especially those of the PCA (Fig. 16).
After the immunoreactive nerves entered the small
pial or intraparenchymal branches via the large
pial branches, they rapidly decreased in number,
and only a few fibres ran spirally or parallel to the
axis of arteriolar branches.
Double immunostainig for NPY and TH showed
that most of the cerebral perivascular NPY nerves
on the BA were immunoreactive to TH. Some of
them showed no immunoreactivity to TH (Fig.
Relative density of NPY nerves in the major cerebral arteries of the greater horseshoe bat and
ICS
Artery IEA ACAI MCA ACA2 AR CCA
PR PCA1PCA2 SCA TA BA
VBS
IAA VAI VA2
Density 2 2 2a weeS 3 ls 2
5 4 4 5 Sm S4. Prd 2
1, very few fibres; 2, few fibres; 3, a moderate number of fibres; 4, numerous fibres; 5, very numerous fibres.
Fics. 8A, B. Fluorescence photomicrographs of the cerebral carotid artery of the greater horseshoe bat with double
immuno-fluorescent staining for NPY (A) and VIP (B). Arrows indicate the thick fibre bundles with both NPY
and VIP immunoreactivities. 100.
Fics. 9-14. Photomicrographs of whole-mounts showing NPY innervation of the major cerebral arteries of the
greater horseshoe bat.
Fig. 9. Middle and anterior cerebral arteries. 67. Fig. 10. Anterior cerebral and
internal ethmoidal arteries. 100. Fig. 11. Posterior ramus, posterior cerebral and superior cerebellar arteries
and terminal branch of basilar artery. X67. Fig. 12. Basilar and internal acoustic arteries (BA, IAA). X67. Fig.
13. Confluence of the basilar and vertebral arteries. x67. Fig. 14 Vertebral artery just behind the entrance of the
cranial cavity. < 100.
830 K. ANDO AND S. ARaAI
iksd=}
Fics. 15, 16. Whole-mounts of pial arterial and arteriolar branches of the posterior cerebral artery (15) and sections
of its small branches perforating into the cerebral cortex (16) of the greater horseshoe bat. Arrowheads indicate
NPY fibers. Fig. 15, 100; Fig. 16, «200.
Fics. 17A, B. Fluorescence photomicrographs of the basilar artery of the greater horseshoe bat with double
immuno-fluorescent staining for TH (A) and NPY (B). Arrowheads indicate NPY nerve fibres without TH
immunoreactivity. 200.
Fics. 18A, B. Fluorescence photomicrographs of a branch from the posterior cerebral artery of the greater
horseshoe bat with double immuno-fluorescent staining for NPY (A) and VIP (B). Arrowheads indicate the axon
with NPY immunoreactivity, but not with VIP immunoreactivity. Arrows indicate the axon with both NPY and
VIP immunoreactivities. 200.
17). By double immunostaining for NPY and VIP, VIP immunoreactivity, while others were im-
it was evident that some of the cerebral perivascu- _uunoreactive to VIP (Fig. 18).
lar NPY axons with terminal appearance had no
Cerebrovascular NPY Innervation in Bats 831
DISCUSSION
There is conclusive evidence that cerebral peri-
vascular noradrenergic and NPY nerves enter the
cranial cavity along the ICA, IEA, and VA, and
mainly come from the ICA [26]. The main origin
of these two types of nerves supplying the cerebral
arterial tree via the above three arteries is the SCG
[7-10]. This neuronal projection seems to be held
the small bats as well: the SICN emanating from
the SCG, which expresses intense NPY im-
munoreactivity as well as formaldehyde-induced
fluorescence for NA, reaches the circle of Willis
along the CCA as several fibre bundles. Fur-
thermore, the presence of one or two fibre bundles
with NPY lying on or along the IEA and the VA is
highly suggestive that cerebral perivascular NPY
nerves also travel along these two arteries.
The present study demonstrated the existence of
many VIP axons within NPY fibre bundles on the
wall of the CCA of the two microchiropteran
species. Since no cell bodies with VIP are found in
their SCG, these VIP fibres seem to be of non-
sympathetic. In the rat, it has recently been
confirmed that cerebral perivascular VIP and cho-
linergic nerves are derived from the three distinct
cranial parasympathetic ganglia of facial or glos-
sopharyngeal nerves, the sphenopalatine ganglion,
the internal carotid miniganglia, and the otic gang-
lion, and that the major source for cerebrovascular
parasympathetic VIP and cholinergic nerves is the
sphenopalatine ganglion [27-31]. In the bent-
winged bat, Ando [26] demonstrated the presence
of microganglia with many VIP and cholinergic cell
bodies in the nerve bundle running in close asso-
ciation to the tympanic part of the SICN. These
microganglia do not emit NA fluorescence, so they
are interpreted as being homolgous with the inter-
nal carotid mini-ganglia located near the carotid
part of the ICA in the rat [32-34] and the monkey
[35, 36]. The microganglion-containing nerve bun-
dle and the SICN are intermingled with each other
to form a nerve bundle consisting of NA fluores-
cent and non-fluorescent axons near the carotid
canal, and some fibre bundles arising from this
nerve bundle enter the cranial cavity along the
ICA. Accordingly, it is most likely that in the
bent-winged bat, the VIP axons within fibre bun-
dles on the CCA, if not all, originate from cell
bodies in these parasympathetic microganglia.
However, no existence of VIP microganglia in any
parts of the tympanic cavity of the greater
horseshoe bat indicates that the VIP axons con-
tained in the corresponding fibre bundles of this
small bat must have their origin at other local or
major cranial parasympathetic ganglia.
Nerves containing NPY but not NA have been
disclosed in the rat VBS [37] after removal of the
SCG. Double staining immunohistochemistry
combined with retrograde tracing techniques has
corroborated in the rat that cerebral perivascular
NPY nerves showing no TH immunoreactivity are
mostly immunoreactive for VIP, and originate
from the cell bodies with both NPY and VIP of the
sphenopalatine ganglion, the internal carotid mini-
ganglia, and the otic ganglion [34]. The appear-
ance of NPY nerves without TH and those with
VIP in the pial arteries has also been noticed in
small bats in the present study, suggesting that
some of the NPY nerves surrounding the bat
cerebral arteries are of non-sympathetic in nature.
The contribution of neurons from the microganglia
in the tympanic cavity of the bent-winged bat to
the cerebrovascular NPY innervation seems to be
ruled out, because no NPY immunoreactivity has
been ascertained within this ganglia. The possibil-
ity that neurons with NPY are present in the
sphenopalatine and otic ganglia of small bats, and
project from there to the cerebral vessels, is still
open.
The most characteristic feature of cerebrovascu-
lar NPY innervation in small bats is that the
density of nerves is distinctly much high in the VBS
than in the ICS, especially throughout the walls of
the PCA to BA. This pattern is quite different
when compared to the general pattern of cerebro-
vascular NPY innervation reported for the rat [38],
gerbil [39], ginea pig [40], cat [13], and man [41]:
the nerves are less densely distributed along the
VBS than along the ICS, with the lowest supply in
the PCA. Thus, the VBS of small bats has the
richest supply of NPY nerves among the mammals
that have been reported up to now. The predomi-
nantly caudal innervation by cerebral perivascular
NPY nerves in small bats results in the preferential
caudal distribution of NPY axons from the fibre
832 K. ANDO AND S. Aral
bundles on the CCA. This is in contrast to the
projection of NPY nerves to the rat cerebral
arterial system from the same neuronal pathway
[26]. The innervation density of NPY nerves in the
respective major cerebral arteries of the bats is
virtually equal to that of noradrenergic, cho-
linergic, and VIP nerves [20, 25], and does not
correspond to the SP innervation which is mainly
confined to the BA and VA, and to the CGRP
innervation which is very sparse in the greater
horseshoe bat and lacking in the bent-winged bat
[42, 43].
There is much evidence concerning the innerva-
tion of small pial and intracerebral arteries by
sympathetic noradrenergic nerves [e.g., 1, 44].
NPY nerve fibres of peripheral origin have also
been demonstrated to associate with intracerebral
arteries and arterioles in the cat [10], whereas
peripheral cholinergic and VIP innervation has not
yet been precisely substantiated for the brain
parenchymal arteries in laboratory mammals. In
our previous studies on the bent-winged bat, the
innervation of intraparenchymal arteries and arter-
ioles not only by sympathetic noradrenergic
nerves, but also by cholinergic and VIP nerves
arising from a peripheral origin has been frequent-
ly encountered in the brain areas where the PCA
gives off many branches [20, 25]. The same was
true for the NPY innervation of these small brain
vessles in the present study.
To date, no information on the functional in-
volvement of NPY that is contained in sympathetic
and parasympathetic nerves has been published for
the bat cerebral circulation. However, in addition
to the predominant development of the VBS in
small bats, the very rich NPY innervation focused
on the bat VBS must be considered in relation to
the vasomotor actions essential for the functioning
of this major arterial system in supplying blood to
their brains during the active and hibernating
seasons. In laboratory mammals, the effects of
NPY on the cerebral circulation, non-adrenergic
and non-cholinergic vasoconstriction, and modula-
tion for the vasoconstriction induced by NA, have
been well documented by pharmacological experi-
ments [11-13]. Therefore, it is possible to predict
that the VBS in small bats may have an important
role in the regulation of local and systemic cerebral
blood flow in correspondence with the changes of
brain metabolic activity characteristic of these
mammals, as a result of such direct and indirect
vasoconstrictor effects of NPY nerves. For more
understanding of the neurogenic control mechan-
isms of the bat cerebral circulation by NPY, it is
necessary to unravel the interaction and competi-
tion between this neuropeptide and other neuro-
transmitters or neuromodulators contained in
cerebrovascular sympathetic, parasympathetic,
and sensory nerves. In this context, marked in-
creases in number of parasympathetic NPY nerves
with VIP [37, 40] and of sensory CGRP nerves [45]
have been established in the cerebral arteries of
the rat and guinea pig after long-term sympathec-
tomy.
ACKNOWLEDGMENTS
This work was supported by a grant from the Kyushu
Sangyo University of Japan.
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ZOOLOGICAL SCIENCE 9: 835-842 (1992)
© 1992 Zoological Society of Japan
Familial Association, Nymphal Development and Population
Density in the Australian Giant Burrowing Cockroach,
Macropanesthia rhinoceros (Blattaria: Blaberidae)
TADAO MATSUMOTO
Department of Biology, College of Arts and Sciences,
The University of Tokyo, Tokyo 153, Japan
ABSTRACT—The Australian giant burrowing cockroach Macropanesthia rhinoceros was studied with
respect to population density, familial association and nymphal development. These, and other field
observations, were made in the eucalypt open woodlands of northeastern Queensland at intervals from
October 1987 to October 1989. Three hundred one nest burrows were excavated in 16 plots (each 8 m X
4m), and 329 adults and lone nymphs (old and middle age) were examined. Thirty-two families, which
consisted of a group of young nymphs together with either an adult pair or an adult female were found in
and around the plots from Nov. 1987 to Mar. 1988. The cockroaches form family groups in the early
spring and close adult female-offspring relationships persist for about half a year. The nymphs reach the
sixth or seventh instar by the autumn. Then they disperse from their natal burrows to make their own.
INTRODUCTION
The cockroaches (Blattaria) are of special in-
terest among the presocial insects because they are
closely related to the Isoptera (termites), all of
whose members are eusocial [1]. The ovovivipar-
ous Blaberidae are frequently subsocial. Roth and
Willis [2] described the familial associations of 15
species of cockroaches, mostly ovoviviparous, that
probably brood their young. Schal et al. [3] discus-
sed the reproductive tactics of cockroach females
and males. Brood care is generally seen in
aggregations of newly-hatched offspring around
their mother, the mother to some extent facilitat-
ing the nymphal aggregation by remaining im-
mobile for varying periods of time [4]. In addition,
some species dwelling inside wood, and feeding on
it, have a monogamous family life. The link
between adults and nymphs in the wood-feeding
cockroaches Cryptocercus (Cryptocercidae) and
Salganea (Blaberidae, Panesthiinae) is long last-
ing, and can last the whole nymphal life [5-8]. The
appearance of a monogamous family structure in
wood-feeding cockroaches and termites is a true
Accepted June 4, 1992
Received April 4, 1992
convergence [9, 10].
The giant burrowing cockroach Macropanesthia
rhinoceros is an ovoviviparous blaberid that dis-
plays brood care and is the largest and bulkiest
blattarid in Australia [11]. The body length of the
largest adult male reaches about 8 cm and the live
weight reaches 30g. However, little information
has been available to date on the ecology in the
field. Only Day [12] notes as follows: “Little is
known of the life history of Macropanesthia, but
some details have been supplied by Mr. W. A.
Henson. The roaches are infrequently observed
during the dry season from March to October.
They burrow quite deeply, about two feet below
the surface of sandy soil in stands of cypress pine
(Callitris sp.). They make a nest of dead leaves,
grass roots, etc., frequently among the pine roots.
The young nymphs rarely appear above ground,
but following rain the adults burrow to the surface,
especially at night”.
The present paper deals with familial associa-
tions, development of nymphs, population density
and other field observations of the giant burrowing
cockroach, M. rhinoceros in the eucalypt open
woodlands of northeastern Queensland. Studies of
the distribution pattern of nests and the material
cycles (carbon and nitrogen) in ecosystem medi-
836
ated by the cockroach will be published elsewhere.
MATERIALS AND METHODS
The giant burrowing cockroach belongs to the
Panesthiinae most species of which are found
primarily in the Indo-Malayan and Australian re-
gions [11]. In the Australian continent, the sub-
family is largely restricted to the eastern part
including species which live in and feed on dead
wood in rain forests (e.g. Panesthia and Ancaudel-
lia) and species which inhabit underground bur-
rows in open woodlands or grasslands. (e.g. Mac-
ropanesthia and Geoscapheus). Almost all of these
taxa are found in Queensland but are comparative-
ly poorly represented in other states. M. rhi-
T. Matsumoto
noceros occurs in rather dry areas around the
tropical rain forests of northeastern Queensland
from Cooktown to Rockhampton along the Great
Dividing Range [11].
The study sites are located in the open eucalypt
woodlands near Smith Creek about 12 km south-
west of a town, Mount Garnet (17.41S, 145.07E,
altitude 680m), and near Uramo, about 10 km
east of the town in northeastern Queensland. The
forest and soil types of Smith Creek and Uramo
are not different. The soil is sandy and not
developed, the thickness of the whole (A) layer
being about 10cm of which litter (Ao) layer is
under lcm. The dry season is from May to
November and wet season is from December to
April and these directly influence the water con-
TasLe 1. Number of nest burrows, cockroaches and social units per plot of 32 m* (8 mx4m) in 16 plots
2) . .
Plot Plies gait Season, Ne. a No. of cockroaches Social unit
No. of survey (temp.) nests Male Female Pair?) Family”
1 Smith 23-28 Oct. 87 Spring 28 15 15 1 1
2 Uramo 32— 2 Nov. (29°C) 18 7 12 0 5
3 Uramo 13-15 Dec. ’87 Summer 20 7 14 1 4
4 Smith 16-18 Dec. (31°C) 29 12 20 3 1
5 Smith 12-13 Mar. 88 Autumn 14 8 0 0
6 Smith 14. Mar. (26°C) 11 4 8 1 0
7 Uramo 15-16 Mar. 18 13 10 3 3
8 Uramo 17. Mar. 26 14 17 3) 2
9 Smith 15-17 Jul. ’89 Winter 13 6 9 2 0
10 Uramo 17-19 Jul. (21°C) 21 11 13 3} 0
11 Uramo 20 Jul. 9 4 5 0 0
12 Smith 21-22 Jul. 16 7 10 1 0
13 Uramo 20- 2 Oct. ’89 Spring 21 11 11 1 0
14 Smith 3— 4 Oct. (26°C) 17 5 14 2 0
15 Uramo 5— 7 Oct. 30 17 14 1 0
16 Smith 8 Oct. 10 5 5) 0 0
Total 301 146 183 24 16
(in average) (18.8) (9.1) (11.4) (1.5) (1.0)
') Mean temperature in the bottom of nest burrows.
season did not vary more than the 1 C precision allowed by the thermometer.
be calculated.
The temperature observed in all nests during the same
Thus no standard deviation could
?) These figures include the number of adults, sub-adults and large nymphs, not include young numphs under
sixth instar.
*») Adult pairs (male and female)
» Families consiting of a group of young mymphs together with an adult pair, and families with an adult female
and young numphs.
Familial Association of Macropanesthia 837
tent of the soil. The sandy soil is compacted in the
dry season and is soft but not sticky in the wet
season. The largest tree measured was about 30
cm in diameter and ca. 25m high. Grasses are
common on the woodland floor and used for
pasture in rainy season. Litter accumulation was
263 g/m? near Smith Creek (n= 11, leaf litter= 124
+76 g, small branch litter=139+82 g) and was
126 g/m? near Uramo (n=11, leaf litter =68 +32
g, small branch litter=58 +52 g).
The field studies and collections were made on
five occasions from October 1987 to October 1989
(Table 1). Sixteen plots (each 8 m x 4 m) and some
areas around the plots were investigated. The
surface of the sandy soil in plots was removed to a
depth of about 10 cm using a scoop, and then, the
entrance holes of burrows were mapped. A trench
about 50cm wide and 50cm deep was then dug
carefully along the nest burrow using shovel for the
heavy work, and a trowel for the finer work. The
burrows descend about 40cm deep in a broad
curve and have a small chamber at the bottom
where the adults live and rear their nymphs. We
opened 301 nest burrows in and around the plots
and examined the cockroaches, food storages,
feces, predators and sometimes guests in the bur-
rows. These items were dried for about 5 days
before being weighed. Some samples of insects
and eggs were kept in 80% alcohol. Eggs in
ovaries and brood pouches were studied by dissec-
tion of females in July and October 1989.
Nest temperature was measured by inserting a
mercury thermometer into the terminal portion of
nest prior to excavating it. The thermometer was
calibrated in 1°C intervals, and all observations
were made during the day.
RESULTS
Nest burrows
Figure 1 shows a schematic presentation of a
nest burrow with an adult pair, nymphs and a
predator (centipede) in spring. The size of the nest
burrow is about 1 m long and 40 cm deep, and it
descends at about a 20° angle. The semi-circular
cross-section of the burrow is 4-15 cm in diameter
and has a plastered wall. The entrance is con-
Fic. 1.
Schematic diagram of a nest burrow of the
Australian giant cockroach Macropanesthia rhi-
noceros with an adult pair, young nymphs and a
predator (centiped) in spring. Leaf litters are trans-
ported by adults from ground surface at night. The
tough adult external skeleton may protect against
centipede attack. (drawn by Y. Ohira)
cealed in raised loose dirt in the dry season. Two
examples of the distribution of nest burrows in the
plots No. 4 and No. 8 are shown in Figure 2. Most
nest burrows are more or less curved. In a few
cases (e.g., Nest No. 30 in Plot No. 4), the burrow
was in the form of a spiral. I did not observe the
case in which two burrows are connected. The
distribution pattern of nest burrows is almost uni-
form. About 10% to 50% of burrows in a quadrat
appeared to be abandoned. In those burrows,
fungi were usually found growing on the remaining
feces and food. The cockroaches prefer litter on
the ground as food, which contains dead leaves,
woods and grasses. A cockroach grasps the food in
its mandibles and transports it walking astride of it.
Foraging activity apparently takes place mostly at
night in the rainy season. We believe the cock-
roaches play an important role in litter turnover
(unpublished).
Population density
Table 1 shows the abundance of nest burrows,
the number of individuals per plot and the sociality
838 T. MATsuMOoTO
Plot 4, Smith Creek
Fic. 2. Two examples of the distribution of nest bur-
rows in the plots No. 4 and No. 8. Most net burrows
are more or less curved. The distribution pattern of
nest burrows is almost uniform. Connection be-
tween two nest burrows is rare. Several (approx.
10%) of burrows in a plot appeared to be aban-
doned.
of groups. In the plot (No. 4) in which nest
burrows were most abundant, there were 29 bur-
rows and 32 large cockroaches. No cases were
seen in which three or more adults occured
together in a burrow. In each plot, a mean of there
was 19 burrows, and 1.5 pairs, 1.01 families were
observed, 9.1 males and 11.4 females live in bur-
rows.
Table 2 summaries the composition of the
groups found in the same burrows and shows the
total number of individuals including young
nymphs which were found with adult pair or adult
females. One hundred forty seven adults, 179
non-adults (sub-adults and old nymphs) and 321
young nymphs were collected from 16 plots. The
sex ratios of adults and non-adults were slightly
lower than 0.5 (males: females=1:1). The sex
ratio of young nymphs was not measured.
Familial composition
Thirty two families consisting of a group of
young nymphs together with an adult pair or an
adult female were observed in and around the
plots from Nov. 1987 to Mar. 1988 (Table 3).
Nymphs are housed in the deepest portions of the
burrows (Fig. 1). Table 4 summarizes number of
eggs in ovaries or the brood sac, and the brood
sized of families. In late October on 1987 (late
spring), eleven families were observed with an
TABLE 2. Composition of the groups collected in 16 plots and number of cockroaches per two or four plots
Plots Plots Plots Plots Plots
No. 1, 2 No. 3, 4 No. 5-8 No. 9-12 No. 13-16
CREO 23-2 Nov. 12-18 Dec. 12-17 Mar. 15-22 Jul. 3028) Octane
1987 1987 1988 1989 1989
Single (adult @) 0 2 1 ‘ 9 14 26
Single (non-adult ¢) 19 12 28 13 20 92
Single (adlult $) 1 2 11 21 20 55
Single (non-adult $) 19 23 15 10 20 87
Pair (1$+1) 1 4 9 6 4 24
Family ( +nymphs) 4 5 5 0 0 14
Family (% 2 +nymphs) 2 0 0 0 0 2
No. of young nymphs* 157 89 75 0 0 321
Total no. of indv.** 206 141 153 65 82 647
Sex ratio ($/7+¢) 0.45 0.35 0.49 0.46 0.44
* Total number of nymphs consisting of a group with an adult pair or an adult female
** adults and non-adults not including young nymphs
TaBLE 3. Family compositions (Adult and young
nymphs) of 32 colonies of M. rhinoceros in and
around the plots
Familial Association of Macropanesthia 839
Number of nymphs
Date of Presence of
Suey adult(s) (instar) (number)
23 Oct. °87 2 Ist instar 29
2 Ist instar 13
30 Oct. 2, 8 lst instar 16
2 Ist instar 17
31 Oct. 9. Ist instar 32
9 Ist instar 32
@ Ist instar 28
Q Ist instar 20
2 Nov. 2 Ist instar 29
4 Nov. Oo, # Ist instar 24
9, 8 Ist instar 22
13 Dec. °87 Oo 2, 3rd instars 20
@ 2, 3rd instars 15
Q 3, 4th instars 13
15 Dec. @ 2, 3rd instars D2
a 3rd instar 21
17 Dec. @ 2, 3rd instars 11
19 Dec. g& 3rd instar 24
2 3rd instar 17
2 3, 4th instars 19 -
2 3, 4th instars 18
@ 3, 4th instars 7
15 Mar. ’88 2 5, 6th instars 19
g 5, 6th instars 17
g 5, 6th instars 12
17 Mar. @ 5, 6th instars 11
o@ 6th instar 17
18 Mar. g 5, 6th instars 18
@ 5, 6th instars 16
Q 5, 6th instars 13
g@ 5, 6th instars 10
2 6th instar 15
average brood size of 22.8. Interestingly, in only
four of thirty two cases were adult males M.
rhinoceros found in familial associations with
nymphs and females. These nests were excavated
in the spring of 1987. In all other cases (n=28),
only adult females were found in familial associa-
tions with nymphs. In middle December of 1987
(summer), nymphs had reached third or fourth
instar and brood size decreased to a mean of
cockroaches 17.0. No adult males were observed
in family groups in the summer. In middle March
on 1988 (autumn and rainy season), nymphs had
reached the fifth or sixth instar and the mean
brood size was 14.8. In late July of 1989 (winter),
no families with young nymphs were observed.
M. rhinoceros is a ovoviviparous cockroach and
new nymphs emerge from the brood sac of female
in spring. All eggs in the ovaries or sacs of each
adult female were removed and weighed as a
group in July 1989. The number of eggs in ovaries
is 23.8 on average as shown in Table 4. The body
length of pre-emergent nymph in brood sac is
about lcm. The newly-emerged cockroaches
might appeared first in middle October (late
spring).
Growth of nymphs
The new nymphs eat leaf litter stored in nest
burrow by the adults. All data on the size of young
nymphs living in nest burrows with adults in late
October 1987, mid December 1987 and mid March
1988 are presented in Figure 3. Nymphs seem to
grow synchronously, and disperse from their nest
burrow in the sixth instar, after which they live a
solitary existence until they become adults. The
body length of adults is about 7-8cm. The size
distribution of pronotum widths of old nymphs and
adults living in a solitary life or forming a pair are
TaBLE 4. Number of eggs in ovaries or brood sac, and brood size of Macropanesthia rhinoceros
Date No. of female Age of rood! Se
of surve SEAS with offsprings offsprings
y Pune Pants Mean+S.D. Range
23 Oct.—4 Nov. ’87 Spring 11 1st instar 23.8+6.7 13-32
13-19 Dec. °87 Summer 11 3—4th instar 17.0+5.1 7-24
12-18 Mar. ’88 Autumn 10 5—6th instar 14.8+3.1 10-19
15-22 Jul. °89 Winter 16 Eggs in ovary 23.8+5.3 13-33
20 Sep.—8 Oct. °89 Spring 17 Eggs in brood sac 22.8+4.6 14-29
840 T. MaATsuMoTO
middle Mar. '88 (fall)
No. of individuals
Width of pronotum (mm)
Fic. 3. The size distribution of pronotum widths of
young nymphs living in nest burrows with adults on
late Oct. ’87, middle Dec. ’87 and middle Mar. ’88.
Nymphs seems to grow synchronously. Instars are
denoted by Roman numerals.
22
204 a
18 Female
= 16
3 14
>
5 12
=eH0)
© 8
(2)
ome
4
2 P
0 IAA
14 16 18 20 22 24 26 28 30 32 34
22
20: Single
b 18 &2 Family
we 16 Pair
3 14
2 12
mo}
&
=
(e)
fe}
z
ONPROAWDS
14 16
i)
iS)
(e%)
aS
18 20 22 24 26 28 30
Width of pronotum (mm)
Fic. 4a, b. The size distribution of pronotum widths of
old nymphs and adults living solitarily, in pairs, and
in familial associations. A few large nymphs and
some adults form pairs, and some adults have young
numphs in their nest burrows. Data from nest
burrows not censused in plots at Smith Creek and
Uramo is included in these figures. A little old-
nymphs and some adults make pair life, and some
adults have young nymphs in their nest burrows.
Instars are denoted by Roman numerals.
shown in Figure 4a, b.
I estimated how many instars the cockroaches
have in nymphal stage on the basis of the size
distribution of pronotum widths in Figure 3 and
4a, b. Sexual dimorphism of M. rhinoceros is not
apparent until the fifth or sixth instar. After this
point the differences in pronatal shape become
clear. A total 597 of predispersal nymphs and 141
free-living nymphs from 16 plots and adjacents
areas were investigated. Nine peaks can be recog-
nized from Figures 3 and 4. The cockroach prob-
ably has nine instars in the nymphal stage.
Nymphs live a solitary life from the seventh to
ninth instar, or in a few cases from the sixth instar
stage. Some adults form pairs which have young
nymphs in the nest burrows as shown Figure 4a, b.
DISCUSSION
Gautier et al. [10] discussed the relationship
between ecology and social behavior in cock-
roaches, and stated the importance of research for
correlations between habitat parameters and social
characteristics. Cockroaches may use any one or a
combination of the following proximate and evolu-
tionary defensive tactics: concealment, evasive be-
haviors, protective or aposemantic colouration,
chemical defence, disturbance sound production,
and fighting [3]. Species of Pycnoscelus, Areniva-
ga, Epilampra, Geoscapheus, Blaberus, Hyporhic-
noda, Eublaberus, and Brysotria cited in Schal et
al. [3] burrow into the substratum during the
inactive period, or in response to disturbance. I
consider that Macropanesthia use apparently the
burrowing behavior for defensive and protective
tactics against natural enemy and severe climate.
Why are the adults of M. rhinoceros so large,
and why do they have a long-lasting family life? To
answer these questions from ecological point of
view, natural enemies comprise the most impor-
tant among many habitat parameters. Field
observations indicated that the main natural ene-
mies are centipedes and large spiders. Both anim-
als were observed frequently in the field, and I
observed a large centipede (Ethomostigmus sp.,
body length is about 18 cm) feeding on a solitaly
nymph in a nest burrow. Centipedes can not eat
adult cockroaches, because the tough adult exter-
nal skeleton protects cockroaches against cen-
tipede attack. This observation was confirmed in a
preliminary manner with a laboratory experiment.
Large spiders (Theraphosidae, body length is ab-
out 4.5cm) were found several times in nest
Familial Association of Macropanesthia 841
burrows where no cockroach was found. It is
possible that nymphs were eaten by the spiders. It
is unlikely that the spiders prey on adult M.
rhinoceros, because these insects are larger and
more heavily armored than the spiders. During
early nymphal stages, when natural enemies in-
vade a nest burrow, adult cockroaches defend
themselves and their offspring. Adults may be
able to block a nest burrow against the enemy by
using its robust body (Fig. 1). I have seen many
large ditches about 20cm deep on the surface of
ground in the field; possibly resulting wild pigs or
marsupials (bandicoots?) which were foraging for
the cockroaches in shallow burrow, but detail are
unknown.
Obviously the shape of M. rhinoceros has
adapted for life in dry sandy areas. The cockroach
has a smooth, flattened body, finger-like spines on
front legs (reminiscent of those of a mole), a
shovel-shaped pronotum, and completely lacks
wings. These aspects of body shape may represent
adaptations for digging in sandy soil. Day [12]
studied on internal anatomy and histology of this
cockroach in comparison with a relatively small
one, Blattella germanica (weight, approximately
0.06 g) and concluded that gravity, diffusion,.and
the area available for secretion and absorption are
not factors limiting the size of M. rhinoceros, and
that the specializations observed in M. rhinoceros
are related to its burrowing habits and food.
Some males of Macropanesthia were observed to
live with a female and first instar nymphs in early
spring (Fig. 4a). This is similar to the monoga-
mous family life observed in xylophagous species
such as Cryptocercus punctulatus, Salganea
taiwanensis and S. esakii [6-8]. But the familial
association of M. rhinoceros does not continue for
long, in that all males leave the families when
nymphs have reached to the second instar. It is
possible that the males search for other females for
mating.
The growth of nymphs is synchronously as
shown in Figure 3. This could be due to the fairly
prolonged dry period in this locality, which limits
the timing of reproduction. And the steady en-
vironmental condition in deep burrow is also im-
portant. I observed that the temperature of bur-
row is warmer than ambient temperature in the
early morning on mid July in 1989 (in mid-winter,
21°C:7 °C), and that of the burrow is cooler than
the ambient temperature in the afternoon on mid
December in 1987 (in mid-summer, 31°C :44°C).
It can be said that the deep burrows buffer the
roaches from severe temperature change. There-
fore, the nymphs can grow synchronously.
However the factors triggering the dispersal of
middle-aged nymphs from their mother’s nest re-
main unclear.
Roth [11, 13-15] divided the Panesthiinae into
five tribes: Panesthiini, Ancaudelini, Salganeini,
Caepariini and Geoscapheini. The first four tribes
all live in and feed on dead wood. All species in
the Geoscapheini, which includes Macropanesthia,
live in burrows and feed on litter from the ground
surface. On the basis of the differences in repro-
ductive behavior observed by Rugg and Rose [16]
and in morphology and habits as noted by Roth
[11, 15], Rugg and Rose [17] believe that the
Geoscapheini represent a sister-group to the other
tribes and should be ranked as a subfamily. I
consider the Geoscapheini, which is composed of
four genera (Macropanesthia, Parapanesthia,
Geoscapheus and Neogeoscapheus), derived from
the ancestor of a xylophagous cockroach similar to
the rain forest Panesthiini, which became secon-
darily adapted for savannah life. The familial bond
of Geoscapheini with adult and offspring in the
ground were strengthend at the same time, when
savannah life was adopted.
ACKNOWLEDGMENTS
The author wishes to express his hearty thanks to Dr.
R. W. Taylor (Division of Entomology, CSIRO) and
Prof. Y. It6 (Nagoya University) for valuable advice
during the whole course of this study, and to Prof. R. H.
Crozier (La Trobe University) and Dr. A. C. Messer
(University of Shizuoka) for critical reading of the manu-
script. Thanks are also extended to Mr. B. Brotherton in
Mount Garnet, and to Drs. H. A. Rose and D. Rugg
(University of Sydney) for giving us information on the
study sites and Macropanesthia, and to Drs. K. Masuko
and Y. Hirono and Messrs. S. Ichitani and Y. Obata
(University of Tokyo) for assisting field studies. This
work was supported by Grant-in-Aid for International
Scientific Research Program (No. 01041029) and in part
for Scientific Research (Nos. 01540543, 02454004,
03269102) from the Ministry of Education, Science and
Culture of Japan.
842
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ZOOLOGICAL SCIENCE 9: 843-857 (1992)
© 1992 Zoological Society of Japan
Notes on the Phylogeny of Various Taxa of the Orthorrhaphous
Brachycera (Insecta: Diptera)
AKIRA NAGATOMI
Entomological Laboratory, Faculty of Agriculture,
Kagoshima University, Kagoshima 890, Japan
ABSTRACT— This paper pursues the recognition and significance of synapomorphic characters for
various taxa, and supplements Woodley (1989) on the interpretation of the phylogeny and classification
of the orthorrhaphous Brachycera. The most plausible phylogenetic relationships of the orthorrhaphous
Brachycera are given at the present state of my knowledge.
INTRODUCTION
Grateful acknowledgment is made to Woodley
[1] for his elaborate treatment of the phylogeny
and classification of the orthorrhaphous
Brachycera, in an acceptable, yet conservative,
way, apart from the use of the names Stra-
tiomyomorpha, Xylophagomorpha, Tabanomor-
pha and Muscomorpha (including Asiloidea etc.).
I now wish to discuss various problems arising
from Woodley’s treatment and furthermore to
express my firm beliefs on the true situation with a
hope that my views (which at present I regard as
beyond doubt) may in their turn be modified by
some future workers.
For essential data and illustrations related to this
paper, other than Woodley, see the following
literature: Colless and McAlpine [2]; Krivosheina
[3]; Mackerras and Fuller [4]; McAlpine, Peter-
son, Shewel, Teskey, Vockeroth and Wood [5];
Nagatomi [6-11]; Nagatomi and Iwata [12, 13];
Nagatomi, Saigusa, Nagatomi and Lyneborg [14-
16]; Stuckenberg [17]; Teskey [18, 19]; Tsacas [20].
From my present knowledge, the most plausible
phylogenetic relationships of the orthorrhaphous
Brachycera are shown in Figs. 1-3.
Accepted April 19, 1992
Received March 12, 1992
GEOLOGICAL AGE OF XYLOPHAGOIDEA
AND TABANOIDEA
Within the Tabanoidea s. lat., the Stratiomyidae
and Tabanidae (which have large numbers of
genera and species) diverge from the central stock
consisting of the Xylophagidae s. lat. and Rha-
gionidae s. lat. I have a strong impression that the
most ancestral extant genera of the Tabanoidea s.
lat. are as follows: Exeretonevra (Australia and
Tasmania), Heterostomus (Chile), Pseudoerinna
(= Bequaertomyia) (N. America and Japan), and
Glutops (N. America, Siberia and Japan). The
first two belong to the Xylophgidae s. lat. and the
last two to the Rhagionidae s. lat.
The Xylophagoidea is certainly older phylogenet-
ically (or more closely related to the nematocerous
ancestor) than the Tabanoidea in the following
more plesiomorphic character states: larval head
strongly sclerotized and non-retractile; female cer-
cus 2-segmented and segment 1 simple (not dilated
posterolaterally or posteroventrally); tibial spur
formula 1:2:2 (or rarely 2:2:2) without excep-
tion in the Xylophagidae s. lat. (from which the
Pantophthalmidae is excluded).
It may be said that the Xylophagoidea shows a
stronger decline in the number of the genera and
species than do the Tabanoidea and Stra-
tiomyoidea.
In the Rhagionidae a number of the Jurassic
fossil records are known and adult female mandi-
bles are present in the Tabanoidea, but these
844 A. NAGATOMI
Stratiomyoidea
Xylophagoidea
Tabanoidea
Stratiomyidae
S Xy lomyidae Nemestrinoidea
Pantophthalmidae Asiloidea
Rachiceridae
1 x Xy lophagidae Vermileonidae
zs Coenomyiidae Apsilocephalidae
Empidoidea
Exeretonevridae
r-T14 Cyclorrhapha
Heterostomidae
Pelecorhynchidae
sO - Stratiomyoidea
Rhagonidae
. Pantophthalmidae
Athericidae
.Xylophagidae s. lat.
-—___ Tabanidae
Rhagionidae s. lat.
Nemestrinidae
wl Athericidae
Acroceridae
Tabanidae
— — Hilarimorphidae
Nemestrinoidea
Bombyliidae Bombyliidae et
Hilarimorphidae
Therevidae
ne other Asiloidea
Scenopinidae
-——— Mydidae
—— Apioceridae
Asilidae
Vermileonidae
t Apsilocephalidae
Empididae
an
Dolichopodidae
Cyclorrhapha
Fics. 1-3. Possible phylogenetic relationships of the orthorrhaphous Brachycera. 2, Simplified from Fig. 1; 3,
modified from Fig. 2 (Pantophthalmidae is treated as the sister group of Stratiomyoidea, and Empidoidea ete. is
omitted). A, Asiloidea; E, Empidoidea; N, Nemestrinoidea; r, Rhagionidae s. lat.; S, Stratiomyoidea; T,
Tabanoidea; Tl, Tabanoidea, s, lat.; X, Xylophagoidea; x, Xylophagidae s. lat.
features are lacking in the Xylophgoidea. How-
ever, these two factors seems to be less significant
in the determination of geological age, because the
fossil records are so incomplete throughout the
Diptera and the presence of adult female madibles
is so sporadic throughout extant Nematocera and
orthorrhaphous Brachycera.
Phylogeny of Orthorrhaphous Brachycera 845
SYSTEMATIC POSITION
Pantophthalmidae
The Pantophthalmidae, which was tentatively
placed in the Xylophagoidea by Nagatomi [9] and
Woodley [1], has been relegated to the Stra-
tiomyoidea by Mackerras and Fuller [4] and Kri-
vosheina [3].
The Pantophthalmidae resemble the Stra-
tiomyidae + Xylomyidae in having the larval head
without paired metachephalic rods and are similar
to the Stratiomyidae (not the Stratiomyidae+
Xylomyidae) in having the female cerci separated
from each other. The Pantophthalmidae are also
more similar in the structures of the male genitalia
to the Stratiomyoidea than to the Xylophgoidea
(see Nagatomi [11]).
Woodley (p. 1376) wrote, “Pupation within a
puparium formed from the larval integument is
unique to the Stratiomyomorpha [Stratiomyidae +
Xylomyidae] within the more primitive Brachycera
and is clearly apomorphic. ------ In my opinion, this
character is the most obvious and conclusive syn-
apomorphy linking two families of brachycerous
flies.” The absence of this conspicuous feature
certainly excludes Pantophthalmidae from the
Stratiomyoidea.
Woodley (p. 1376) also wrote as to the position
of Pantophthalmidae, “Further evidence is neces-
sary before their relationships to other primitive
Brachycera can be elucidated.” In any case, the
Pantophthalmidae seems to be intermediate in
position between the Xylophagoidea and Stra-
tiomyoidea and should be relegated to one of
them.
Exeretonevridae (Exeretonevra) and Rhagionidae
(Austroleptis)
Colless and McAlpine ([2] p.755) wrote,
“Typical soil-dwelling xylophagid larvae (see e.g.,
[5]) have been found in Tasmania (M. Williams,
pers. comm.), but no adults have been reared. It
seems likely that they will be found to belong to
some genus currently placed in another family.
Atherimorpha and Exeretonevra, currently in Rha-
gionidae and Nemestrinidae respectively, seems
likely candidates. In any case, we propose on
general grounds to transfer to latter genus to this
family [=Xylophagidae]. Despite some resembl-
ance to Nemestrinidae in wing venation, Exere-
tonevra is clearly misplaced there; but Nagatomi’s
(1977) erection of a monotypic family seems to us
premature. Adults of Exeretonevra occur in high-
land areas from northern N. S. W. to Tas., and
may be locally common on vegetaion near streams.
Their flight is weak and slow, and they appear
reluctant to fly.”
It is unlikely that Atherimorpha, which is more
closely related to Rhagio than to Chrysopilus (see
Nagatomi [10]; Nagatomi and Nagatomi [21]), has
typical xylophagid-type larva. It is almost certain
that the larvae in question belong to Exeretonevra.
If so, Exeretonevra would prove itself to be a
member of the Xylophgidae s. lat.
Austroleptis differs from the members of Rha-
gionidae by the combination of following ple-
siomorphic and apomorphic character states: face
flat and shallow dish-like (plesiomorphic); female
sternum 9 present (plesiomorphic); female cercus
1-segmented (apomorphic).
Colless and McAlpine ((22] p. 701; [2] p. 753)
wrote, “Austroleptis rhyphoides has been reared
from rotting wood.” Xylophagid larvae differ
markedly from rhagionid larvae. It is probable
that the larva of Austroleptis does not fall into the
xylophagid type. If so, Austroleptis would be most
closely related to the Rhagionidae within extant
families of the lower Brachycera, granting that
Austroleptis is treated as representing an inde-
pendent family.
Pelecorhynchidae (Glutops)
Glutops was placed in the Pelecorhynchidae
(Teskey [18, 23]; Woodley), in the Rhagionidae
(Nagatomi [8, 9]) or in the Glutopidae (Kri-
vosheina [24, 3]).
Various views on the phylogenetic relationships
between the families of Tabanoidea (Pelecorhyn-
chidae, Rhagionidae, Athericidae, Tabanidae or
Glutopidae) are shown in Figs. 4-6.
Stuckenberg [17] and Nagatomi [9] considered
the larval mandible with a poison canal as one of
the synapomorphic characters for the Tabanidae +
Athericidae. Woodley took this character as the
evidence which supports the monophyly of Glu-
846 A. NAGATOMI
Pelecorhynchidae
Rhagionidae
(including Glutops)
Athericidae 4
Tabanidae
Rhagionidae
Pelecorhynchidae
(including Glutops)
Athericidae 5
Vawciebibes
Fics. 4-7.
Krivosheina [3]; 7, prepared for the present paper.
tops (as the representative of Pelecorhynchidae) +
Athericidae + Tabanidae. However, it can be im-
agined that the character state above is due to
either convergence or symplesiomorphy.
Krivosheina [24] erected a monotypic family
Glutopidae and later, Krivosheina [3], interpreted
its phylogenetic position as shown in Fig. 6 on the
following basis: “The tracheal branches of the
larvae are fused posteriorly and the caudal spira-
cles are small, point-like in Tabanidae and Gluto-
pidae or are lacking in Athericidae” (see figs. 8-13
in Krivosheina [3]). However, this similarity is
undoubtedly due to the result of convergence in
adapting to aquatic life.
I am convinced that Pelecorhynchus and Glutops
are more closely related phylogenetically to the
Rhagionidae than to the Athericidae + Tabanidae.
The definitive synapomorphic character for Pele-
corhynchus + Glutops + Pseudoerinna+ Rhagioni-
dae is as follows: segment 1 of the female cercus is
dilated posterolaterally or posteroventrally,
although in genera of the subfamily Spaniinae, the
bases of the cerci are more widely separated and
the posterolateral part of segment 1 is usually not
dilated, undoubtedly due to the result of secondary
development or reduction (see figs. 12-25 in Naga-
tomi and Iwata [12]).
Thus, judgment varies with the supposed syn-
apomorphic character adopted such as (1) larval
mandible with poison canal, (2) caudal spiracles
small and point-like, and (3) segment 1 of female
cercus dilated posterolaterally. Character (3) can-
.Glutopidae
Athericidae
Tabanidae 6
Pelecorhynchidae
Rhnagionidae
Pelecorhynchidae
(including Glutops)
——— Rhagionidae
Athericidae 7
Tabanidae
Proposed phylogenetic relationships of Tabanoidea. 4, After Nagatomi [9]; 5, after Woodley [1]; 6, after
not be rejected, because various features of Glu-
tops are so similar in both adult and larval stages to
those of the Pelecorhynchidae + Rhagionidae and
are so sharply different from those of the Atherici-
dae+Tabanidae. The Athericidae+Tabanidae
has definitive synapomorphic characters which are
absent in the Pelecorhynchidae and Rhagionidae
and which are discussed in the forthcoming sec-
tion.
Mackerras and Fuller ([4] p. 29) concluded that
“Thus, on its larval characters alone, Pelecorhyn-
chus would be classified without hesitation as a
Rhagionid, and the question of its relationship
with the Tabanidae or the Stratiomyoid families
[including Xylophagidae s. lat.] would hardly
arise.” For the basis of this fundamental conclu-
sion, see Mackerras and Fuller ([4] p. 28-29). It
must be confirmed whether or not Pelecorhynchus
has the larval mandible with a poison canal, which
is present in Glutops, Athericidae and Tabanidae.
Mackerras and Fuller ({4] p. 29) wrote of the larva:
“Pelecorhynchus has no well-marked relationships
with the Tabanidae, differing from all known
Tabanids in being rigid and incapable of contrac-
tion, and in the absence of pseudopods striations,
and rugose plates. Also the mouthparts of Taba-
nus are highly specialized and have no oral aper-
ture, the food being taken in through the mandibu-
lar pore.”
Stuckenberg ([17] p. 669) wrote, “In Rhagio the
distal portion of the mandible has an internal
channel but this does not open to the exterior
Phylogeny of Orthorrhaphous Brachycera 847
apically, and there are no poison glands (Roberts,
1969: 385).” Regrettably, the nature of this chan-
nel is unknown to me.
Clearly, further studies should be made on the
mandibular pore in the Tabanoidea.
Concerning the family position of Glutops, I
must change my previous idea (Nagatomi (8-11,
25]), admitting that the similarity of the larval
general body form between Glutops and Pele-
corhynchus is not due to convergence or symple-
siomorphy but is due to synapomorphy. It seems
to me in the present instance that the synapomor-
phy is the strongest probability in this case. Here,
I must transfer Glutops from the Rhagionidae to
the Pelecorhynchidae, following Teskey [18, 23]
and Woodley [1], though I believe that the sister-
group of Pelecorhynchidae is not the Athericidae
+Tabanidae but the Rhagionidae (see Fig. 7).
Vermileonidae
Nagatomi ef al. [15] offer a hypothesis that the
Vermileonidae is a sister group of the Apsi-
locephalidae + Empidoidea+ Cyclorrhapha on the
following basis: the structure of the larval head;
the shape of antennal segment 3 and style; the
isolated position of the Vermileonidae within the
taxa of the Tabanoidea s. lat.
Tsacas [20] studied in detail the structure and
function of the larval head and mouthparts in
Rhagio scolopaceus, Chrysopilus auratus and Ver-
mileo vermileo, and concluded that “Un fait est dés
maintenant acquis, c’est que les Rhagionidae
présentent des affinités dune part par les Rha-
gioninae avec les Tabanidae et, d’autre part, par
les Vermileoninae avec les Asilidae et Empidi-
formia.”
I believe there are difficulties which rule against
the above hypothesis.
(1) There are some common characters in the
Stratiomyidae, Xylomyidae, Pantophthalmidae
and Vermileonidae as follows: no dorsal and ven-
tral plates enclosing aedeagus; larval head without
metacephalic rods.
(2) The paired metacephalic rods (which are
absent in the Vermileonidae) are present in the
Empidoidea.
(3) Vermileonidae differs considerably from
Apsilocephalidae, Empidoidea, and Cyclorrhapha
in many external characteristics.
(4) The male genitalia of Apsilocephalidae,
which resemble those of Asilidae, differ so much
from those of Vermilionidae.
These points are now further elaborated.
(1) In Vermileonidae, the larval head is com-
pletely retractile within the thorax; the female
cercus (which may be 2-segmentee as a basic plan)
is unique in shape and autapomorphic for this
family; and so on. The monophyly of Stratio-
myoidea-+ Pantophthalmidae + Vermileonidae is a
weak possibility.
(2) The paired metachphalic rods arose inde-
pendently in the Asiloidea (where the rod is not
paired but single) and Empidoidea, respectively,
as discussed by Woodley (pp. 1386, 1388, 1391).
(3) The Vermilenonidae is situated near the
orthorrhaphous Brachycera stem and has many
plesiomorphic characters.
(4) The transition in evolutionary line of the
male genitalia is not definite among the Ver-
mileonidae, Apsilocephalidae and Empidoidea,
although well-developed and more complicated
surstyli in the Apsilocephalidae show such transi-
tion towards the Empidoidea. However, discrep-
ancy in the structure of the male genitalia between
Vermileonidae and Apsilocephalidae may be due
to the fact that these two taxa are relics of antiquity
and distantly related to each other.
I realize some of these arguments may be re-
garded as subjective.
Nemestrinoidea
Verrall [26], Malloch [27], Colless and McAI-
pine [22, 2], etc. placed the Nemestrinidae and
Acroceridae in the Tabanoidea s. lat. and Bomby-
liidae in the Asiloidea. In the Nemestrinidae and
Acroceridae, the pulvilliform empodium is present
and the macrosetae on the body are absent. These
two plesiomorphic character states seem to be
generally underestimated in the present day under-
standing of the phylogeny of the orthorrhaphous
Brachycera. The presence or absence of charac-
ters mentioned above often varies with genus or
family within the same family or superfamily, that
is, (1) the presence of pulvilliform empodium in
some Asilidae (see fig. 4 in Oldroyd [28]), some
Empididae and some Dolichopodidae; (2) the ab-
848 A. NAGATOMI
sence of macrosetae in the Scenopinidae, Mydi-
dae, Hilarimorphidae and some Bombyliidae; (3)
the presence of macrosetae in Atherimorpha (Rha-
gionidae) from South America; (4) the absence of
the empodium in some Acroceridae. However,
(1), (3) and (4) above undoubtedly occurred
secondarily. So, these two character states should
be reassessed. If they are significant to some
degree in elucidating phylogeny, the treatment by
several workers mentioned above should be re-
vived.
Woodley placed the Nemestrinoidea in his Mus-
comorpha (Nemestrinoidea-+ Asiloidea [including
Bombyliidae]+ Empidoidea+Muscoidea) on the
follwing three bases: (1) Antennal flagellum with
only four (or fewer) flagellomeres; (2) Tibial spurs
lost; (3) Female cerci one-segmented. However,
these three characters very often occurred second-
arily in many taxa of Tabanoidea s. lat., and it is
possible that they became entirely so in the
Nemestrinidae and Acroceridae within the Taba-
noidea s. lat.
No definite synapomorphic character occurs for
the Tabanoidea + Nemestrinoidea and the position
of Nemestrinoidea is vague, but I prefer to place
the Nemestrinoidea in the Tabanoidea s. lat. based
on negative evidence which is discussed again in
the forthcoming section.
Bombyliidae
Which of the Nemestrinoidea and Asiloidea is
the nearer relative of the Bombyliidae? The
Bombyliidae is related to the Nemestrinoidea by
having the following characters: larval head with-
out metacephalic rod; larva parasitic, with hyper-
metamorphosis. On the other hand, the Bomby-
liidae resembles the Asiloidea in having the follow-
ing apomorphic characters: larval posterior spira-
cles located on the apparent penultimate abdomi-
nal segment and more widely separated from each
other; empodium bristle-like; body very often with
macrosetae; female abdomen very often with acan-
thophorites.
Woodley (p. 1385) wrote, “First instar bomby-
lid larvae are active, whereas later instars are
grub-like after they have located and infested their
host. The only other Brachycera outside of the
Muscoidea | =Cyclorrhapha] that are parasitic are
the Nemestrinoidea, treated above. The two
groups have apparently evolved along these lines
convergently, as other evidence suggests the place-
ment of the bombyliidae in the Asiloidea. ------ the
first instar larvae (of Bombyliidae) bear a resem-
blance to those of Therevidae and Scenopinidae,
especially in having long lateral setae on the thorax
and a small, exerted head capsule (Verrall 1909:
fig. 64). +--+ I therefore conclude that parasitism
and hypermetamorphosis are autapomorphic for
the Bombyliidae.”
I have been privileged to see an unpublished
manuscript by D. K. Yeates and M. E. Irwin, who
write, “Thus it is possible that the most plesiomor-
phic larvae in the Bombyliidae (e.g., Glabellula of
the subfamily Mythicomyiinae; see Andersson
[29]) are predatory and lack hypermetamorpho-
sis.”
The immature stages are unknown in the Hilar-
imorphidae which is thought to be the nearest
relative of Bombyliidae. Furhter studies are des-
perately needed on the larval stages of primitive
Bombyliidae.
At present, I would like to place the Bomby-
liidae in the Asiloidea, following Woodley. After
all, the absence of a pulvilliform empodium has led
to the placement of Bombyliidae in the Asiloidea.
SYNAPOMORPHIC OR AUTAPOMORPHIC
CHARACTER
Tabanoidea s. lat. (including Nemestrinoidea)
No definite autapomorphic character state is
found for the Tabanoidea s. lat. “Accordingly,
present knowledge does not provide support for
the Tabanomorpha sensu Hennig [=Tabanoidea s.
lat.] as being a monophyletic taxon” (Woodley, p.
1373):
The component families are united with one
another by having the following plesiomorphic
characters: pulvilliform empodium; no mac-
rosetae; no acanthophorites.
Except for the Nemestrinoidea, most Stra-
tiomyidae and some Rhagionidae, the component
families have bare tibial spurs apparently derived
from the nematocerous ancestor. Nevertheless,
bare tibial spurs would become synapomorphic for
Phylogeny of Orthorrhaphous Brachycera 849
the Tabanoidea s. lat. in relation to the Nema-
tocera, if pilose tibial spurs are a basic plan for the
Nematocera. However, the presence or absence of
pile on spurs may possibly be too slight for cladistic
significance.
Except for the Nemestrinoidea, Athericidae,
Rachiceridae, many Rhagionidae, many Stra-
tiomyidae, and some Vermileonidae, the antennal
flagellum is 8-segmented. This character, which is
definitely a basic plan for the Tabanoidea s. lat., is
plesiomorphic in relation to the Asiloidea, Empi-
doidea, and Cyclorrhapha but synapomorphic for
the Tabanoidea s. lat. in relation to the Nema-
tocera.
Stratiomyoidea+ Xylophagoidea
Woodley (p. 1375) wrote, “:-- there is at present
no conclusive evidence that the three families
[Stratiomyidae, Xylomyidae and Xylophagidae s.
lat.] form a monophyletic group.” Indeed, it is so.
The two superfaimilies are united with each
other by the following plesiomrphic character
states: larval head strongly sclerotized and non-
retractile; female cercus 2-segmented, with seg-
ment 1 simple (not dilated posterolaterally or
posteroventrally).
The non-retractile larval head of Stratiomyoidea
+ Xylophagoidea differs from that of the Nema-
tocera by the head capsule elongated posteriorly
into thorax (Woodley, p. 1372), and in this respect
it becomes synapomorphic character for the Stra-
tiomyoidea+ Xylophagoidea in relation to the
Nematocera, although a pair of metacephalic rods
are present in the Xylophagidae s. lat. but absent
in the Stratiomyidae, Xylomyidae and Pantoph-
thalmidae, and a non-retractile head is also seen in
the Therevidae+Scenopinidae within the Asi-
loidea.
Except for the Xylomyidae and several Stra-
tiomyidae, the clypeus is “flattened and shallow
dish-like (=plate like), i.e., with the margin more
or less turned up and continued around below the
palpus and proboscis (=FP)” (Nagatomi [30] p.
397), as an undoubted basic plan for the Stra-
tiomyoidea+ Xylophagoidea. I still believe that
this character is derived from the nematocerous
ancestor. Nevertheless, the detail of this character
may possibly be different from that of Nema-
tocera. If so, FP-face would be synapomorphic in
relation to the Nematocera or even autoapomor-
phic for the Stratiomyoidea+ Xylophagoidea.
Comparative studies throughout the Nematocera
are necessary to establish the validity of this
hypothesis.
For the monophyly of the Stratiomyoidea+
Xylophagoidea, there is one piece of circumstan-
tial evidence, i.e., the presence of intermediate
forms, namely, the Beridinae, Chiromyzinae,
Parhadrestiinae, Xylomyidae and Pantophthalmi-
dae that link the specialized Stratiomyidae with the
Xylophagidae s. lat.
Tabanoidea+ Nemestrinoidea
There is one common character for the Taba-
noidea+ Nemestrinoidea, i.e., the larval head is
completely retractile within the thorax and has no
metacephalic rod, although the Vermileonidae and
Bombyliidae share these characters.
The Tabanoidea and Nemestrinoidea (as well as
some primitive Bombyliidae) are linked by the
following plesiomorphic character state: larval
posterior spiracles are present on the last abdomin-
al segment and are more closely located to each
other. |
Woodley (p. 1384) wrote, “All larvae of Asi-
loidea [including most of the Bombyliidae] have
the posterior spiracle located in the apparent
penultimate segment of the abdomen, directed
more or less laterally (figs. 37.23, 38.11, 40.28,
42.76-77). This character state is not found in the
Nemestrinoidea or in the other three infraorders of
Brachycera. In the Scenopinidae and Therevidae,
a more derived state is found, in which the pos-
terior spiracle is apparently in the antepenultimate
abdominal segment.”
For the systematic position of the Vermileoni-
dae and Bombyliidae, see preceding section.
Rachiceridae + Xylophagidae
There is one character state for the Rachiceridae
+Xylophagidae, i.e., the cerci are fused for
almost the whole length of segment 1, tergum 9 is
longer than wide, and tergum 10 is absent or
reduced to a pair of indistinct sclerites (see figs. 4—
6 in Nagatomi and Iwata [12]; tergum 10 is well
developed in the Coenomyiidae and Heterostomi-
850 A. NAGATOMI
dae but less developed or possibly absent in the
Exeretonevridae). This character state will be-
come autapomorphic, when commonly seen.
Judging from illustrations (figs. 7-8 in Webb [32]),
the female cerci of Rachicerus obscuripennis are
widely separated. The examination of more spe-
cies is needed in this respect.
There are some common adult characters in the
Rachiceridae and Xylophagidae, i.e., vein Rs ends
at or very near the wing tip; alula is practically
absent (margion of alula straight or nearly so);
eyes in both sexes are widely separated. However,
none of them can possibly be defined as a synapo-
morphic character.
If monophyly of the Rachiceridae + Xylophagi-
dae is true, synapomorphic characters may be seen
in the larval stage.
James [31] prepared a key to genera of the
Xylophagidae s. lat. based on the larva as follows:
“Head capsule at least three times as long as
broad. Thoracic segment with sclerotized plates
lorsallhy (tyes. 5 113))) conesseossrocosonoonoce ensescquasceaae
ual eed ES ee Rachicerus and Xylophagus
Head capsule not more than twice as long as
broad. Thoracic segment not sclerotized dorsally
(ir UID) Secesonbgaandscusondsoddoneoouconuass Coenomyia.”
Two character states in the key will be synapo-
morphic or autapomorphic for the Rachiceridae +
Xylophagidae beyond doubt, when commonly
seen. The two characteristics quoted under
Coenomyia are applicable to Arthropeas sibirica
(see Krivosheina [33]) and Dialysis fasciventris
(see Webb and Lisowski [34]) of the Coenomy-
lidae.
There are some conspicuous autapomorphic
characters for the members of Rachiceridae, i.e.
The antennal flagellum is over 10-segmented and
either pectinate or serrate; apical portion of the
aedeagus consists of 2 tubes in vertical plane and
there are no dorsal and ventral plates enclosing the
apical portion of aedeagus. There is also a possible
autapomorphic character, i.e., the dorsal part of
larval prothorax has an assemblage of sclerotized
amoeba-shaped dots (see figs. 13-14 in James
[31].
For the members of Xylophagidae, there is an
evident autapomorphic character, i.e., the last
antennal segment is not pointed but rounded. This
character is unique within those Tabanoidea hav-
ing 8-segmented antennal flagellum. A possible
autapomorphic character is also seen: the sclero-
tized dorsal plates on the larval thorax are exten-
sive in area and not consisting of amoeba-shaped
dots (see fig. 12 in James [31]).
Coenomyiidae
There is one clear autapomorphic character
throughout the genera of Coenomyiidae, 1.e., the
mid-ventral part of the fused gonocoxites has a
large desclerotized patch which is transversely
elongate and which is distinct from the posterior
margin of the fused gonocoxite (very often except-
ing mid-posterior channel leading to the posterior
margin). This peculiar membranous patch dis-
appears in Dialysis kesseli and Napemyia illinoen-
sis (after Webb [35, 36]) certainly due to secondary
reduction.
Apart form the state of apomorphy or ple-
siomorphy, the male genitalia of Coenomytidae
differ strikingly in many respects from those of
other taxa of Xylophagidae s. lat., namely,
Rachiceride, Xylophagidae, Exeretonevridae and
Heterostomidae, but resemble those of Rhagioni-
dae and Glutops (Pelecorhynchidae) of Taba-
noidea (see Nagatomi [11] p. 155 who wrote, “the
genera of Coenomyiidae, which are almost identic-
al with one another in male genitalia, may easily be
separated from each genus of Rhagionidae, which
may be characteristic in the details of male geni-
talia.”).
The palpus is 1-segmented in the Coenomyiidae
except for some individuals of Arthropeas species
with 2 segments (Nagatomi [37]). The 1-
segmented palpus is nuique within the Xylophagi-
dae s. lat.
The larval anal segment and the sclerotized
dorsal plate (surrounding the posterior spiracles)
may possibly be relatively larger in the Coenomy-
iidae than in the Rachiceridae and Xylophagidae
(see figs. 11-12 in James [31]). If it is constantly
recognized, this character state will become syn-
apomorphic for the Coenomyiidae.
The genera of Coenomyiidae are easily sepa-
rated from those of other families which resemble
the former in appearance (see Nagatomi [6-8}]).
Nevertheless, to find a synapomorphic character is
Phylogeny of Orthorrhaphous Brachycera 851
unexpectedly difficult for the Coenomyiidae. One
reason is certainly that the state of apomorphy or
plesiomorphy is hard to determine in the structure
of male genitalia and the details of wing venation.
However, the principal reason should be attribut-
able to a certain type of primitiveness possessed by
the Coenomyiidae. Some primitive characters
must be specialezed in order to survive for a long
time, but the Coenomyiidae has achieved this
without a strong specialization.
Exeretonevridae + Heterostomidae
There are two characteristics: ocellar triangle
much longer than wide; upper part of clypeus
protruded forward to some extent. Unless the
states are the result of convergence, these two
character states become synapomorphic for the
Exeretonevridae + Heterostomidae, which differ
also from the Coenomyiidae by having two ple-
siomorphic features: palpus 2-segmented; meta-
pleuron bare.
The following are autapomorphic characters for
the Exeretonevridae: “vein R>,3 curved upward at
apical portion but ending far beyond apex of R;; a
crossvein present between veins R>,3 and R4+Rs;
directly arising from discal cell and vein Rs ending
distinctly before wing tip; vein M, ending before
and M> ending far beyond wing tip; vein M3
meeting with vein M, before wing margin; poste-
rior callus (near metapleuron) with a flat elevation
which is elliptical, wider than long and minute
pilose; abdominal tergum 1 with a pair of bare,
elliptical, flat, caudal callosities which are wider
than long” (Nagatomi [9]).
On the other hand, only one autapomorphic
character is seen for the Heterostomidae: the
second palpal segment is much widened. This
character state 1s also seen in the Xylophagidae,
where it probably evolved independently.
The monophyly of the Exeretonevridae+
Heterostomidae + Coenomyiidae is uncertain, be-
cause no definite synapomorphic character has
been detected. Only through the general appear-
ance, may it be said that the Exeretonevridae +
Heterostomidae is more closely related to the
Coenomyiidae than to the Rachiceridae+
Xylophagidae.
Pelecorhychidae + Rhagionidae
As discussed in the preceding section, there is
one definite autapomorphic character for the Pele-
corhynchidae + Rhagionidae, that is, segment 1 of
the female cercus is dilated popsterolaterally,
although this dilation disappears in Austroleptis
(Austroleptinae), Ptiolina and Spania (Spaniinae).
For the Pelecorhynchidae, consisting of Pele-
corhynchus and Glutops, there is an apparent
autapomorphic character, i.e., “larva with stout
spines laterally on labrum and apically on maxilla”
(Woodley p. 1381) (see Mackerras and Fuller [4];
Teskey [18]).
Woodley (p. 1380) wrote, “Glutops and
Pseudoerinna are more similar in general appear-
ance to Rhagionidae than is Pelecorhynchus, but,
based on the extreme similarity of the general
body form of larvae of Pelecorhynchus and Glu-
tops (Teskey 1970) and on the difference between
these and known rhagionid larvae, the two are
likely closely related. The larvae are not known
for Pseudoerinna.” | quite agree with his statement
quoted above, to which some supplementary notes
are here appended.
For Glutops-+ Pseudoerinna+ Pelecorhynchus,
there is apparent synapomorphic characters in
female terminalia, 1.e., segment 1 of cercus situ-
ated near base of sternum 10; tergum 8 including
anterior membranous part much longer than wide;
tergum 10 absent or reduced to a pair of indistinct
sclerites. The female terminalia of Glutops and
Pseudoerinna differ from those of Pelecorhynchus
in two ways: segment 1 of the cercus has a strong
posterolateral process and tergum 8 is more exten-
sively sclerotized, showing the apparent monophy-
ly of Glutops and Pseudoerinna.
The female terminalia of Rhagionidae are
broadly divided into the generalized and special-
ized forms, of which the former has the following
characters: segment 1 of cercus dilated postero-
laterally and situated near apex of sternum 10;
tergum 10 well developed. On the other hand, the
specialized forms are given below:
Austroleptis (Austroleptinae): cercus 1-seg-
mented, not dilated posterolaterally, and situated
near base of sternum 10; tergum 10 reduced to a
pair of small sclerites. Ptiolina and Spania (Spa-
852 A. NAGATOMI
niinae): cercus not dilated posterolaterally; paired
cerci more widely separated at bases; tergum 10
short in Ptiolina and absent in Spania. Spaniopsis
(Spaniinae), and Atherimorpha (Rhagioninae) (af-
ter Mackerras and Fuller ([4] p. 15): segment 1 of
cercus more widely separated (at least in Spaniop-
sis), with a distinct inner section but dilated poste-
rolaterally; tergum 10 well developed in Atheri-
morpha and absent in Spaniopsis.
For a discussion of the female terminalia of the
lower Brachycera, see Nagatomi and Iwata [12,
13].
Pelecorhynchus is very similar to Glutops,
Pseudoerinna and many genera of Rhagionidae in
the structure of female terminalia, but differs
markedly from the latter in the structure of the
male genitalia (see Mackerras and Fuller [4];
Nagatomi [11]).
The external adult characters of Pelecorhynchus
are also conspicuously different from those of
Glutops, Pseudoerinna and all known genera of
Rhagionidae by having the following autapomor-
phic characters: cheek (below palpus) with a large
bulbous protuberance which has many long erect
hairs and which varies in size and shape with sex
and species; palpus shorter, broadly cylindrical,
not tapering toward apex, and with an apical pit or
with a transverse section; 2nd submarginal cell
shorter and much wider; (see Nagatomi [9]).
Glutops differs from Pseudoerinna in the follow-
ing apomorphic characters: tibial spurs 0:2:1 (not
1:2:2); side of face and facial swelling large and
produced forward. The apomorphic characters of
Pseudoerinna in relation to Glutops are as follows:
metapleuron (except lower border) wholly pilose
(not bare); 2nd submarginal cell shorter and wider.
See Nagatomi and Saigusa [38] and Nagatomi [10].
In short, the synapomorphic characters for the
Pelecorhynchidae including Pseudoerinna (whose
larva is unknown) and Glutops are as follows:
“larva with stout spines laterally on labrum and
apically on maxilla” (Woodley) already quoted;
larval body long (in relation to the Rhagionidae),
smooth and without creeping welts; three features
in female terminalia already mentioned.
No definite synapomorphic character has been
found for the Rhagionidae, as long as the Pele-
corhynchidae is treated as an independent family.
I believe that several genera of Rhagionidae,
whose larvae are unknown, are not so different in
the immature stage from Rhagio, Chrysopilus,
Symphoromyia (Rhagioninae) and Ptiolina (Spa-
niinae), whose larvae have a common character,
that is, “terminal segment with lobes or tubercles
of various form surrounding spiracles” (see figs.
16, 18-19, 20-21 in James and Turner [39]), and
the spiracular disc is concave or cleft. This charac-
ter state may be unique for the Rhagionidae within
the Tabanoidea.
Athericidae + Tabanidae
Stuckenberg [17] first established the monophyly
of the Athericidae + Tabanidae.
The most reliable synapomorphic character for
the Athericidae+Tabanidae is the presence of
long aedeagal tines which are also seen independ-
ently in the genus Bolbomyia of Rhagionidae (see
Nagatomi [11]). For the origin of aedeagal tines,
see Nagatomi [40] who discussed the homologies
of several characters along and behind the
aedeagus in the lower Brachycera.
The male genitalia of Dasyomma, the most
primitive genus of Athericidae, are very similar to
those of Tabanidae. Nagatomi ({11] p. 101) wrote:
“Tergum 9 not divided into a pair of sclerites and
its anterior margin not concave ......... Athericidae
Tergum 9 divided or not divided into a pair of
sclerites, and in the latter case its anterior margin
deeply concave:...s...2:.-2o2.009. eee Tabanidae.”
Nagatomi ([{11] p. 145) also wrote, “It appears
that the posterior part of aedeagus is present or
longer in Tabanidae but absent or shorter in
Athericidae.”
The similarity of female terminalia between
Athericidae and Tabanidae may also represent the
monophyly of these two families, in which the
ovipositor is not telescoped; cercus 1-segmented,
generally roughly as wide as long; tergum 8 much
smaller than tergum 7; terga 7-8 much wider than
long; intersegmental membrane between terga 7-8
short or hardly visible (see Iwata and Nagatomi
[41] and Nagatomi and Iwata [12]).
Nagatomi and Iwata [12] wrote, “Athericidae
are very similar in shape of female terminalia to
Pangoniinae of Tabanidae and are difficult to
distinguish from the latter. But in Athericidae
Phylogeny of Orthorrhaphous Brachycera 853
tergum 9 is larger than in usual Pangoniinae, and
mid-distal part to genital furca protrudes forward
(toward base of abdomen), allthough in shape of
genital furca some Pangoniinae resemble some-
what Atrichops.”
It is still inconceivable to me that the Pele-
corhynchidae (including Glutops and Pseudoerin-
na) is the sister group of Athericidae + Tabanidae,
because the male and female genitalia and the
larval body of the former differ so much in many
respects from those of the latter.
Vermileonidae+ Apsilocephalidae + Empidoidea+
Cyclorrhapha
There is one synapomorphic character for the
above taxa, 1.e., “antennal segment 3 is rounded,
triangular or pyriform, and the style is needle-like
or straight, tapering apically and pointed” (see
figs. 1-46 in Nagatomi et al. [15]). However, there
are sO many deviations from the typical scheme
and it is difficult to define the shape of antennal
segment 3 and style precisely. Nevertheless, I
believe that this character state is common
throughout the above taxa as a basic plan.
The larval head of Vermileonidae resembles
that of some Empidoidea (see Tsacas [20]; Teskey
[19]). Unfortunately, the immature stages of Apsi-
locephalidae are unknown.
The monophyly of the Apsilocephalidae +
Empidoidea is uncetain, because no definite syn-
apomorphic character has been found. The sister
group of Apsilocephalidae is probably the Empi-
doidea+ Cyclorrhapha. For the diagnosis of Apsi-
locephalidae, see Nagatomi et al. [14, 16].
For the monophyly of the above taxa, there is
one piece of circumstantial evidence, i.e., there is
a big mrphological gap, on the whole between the
Asiloidea and Empidoidea, apart from the pres-
ence of acanthophorites which has a scattered
distribution in the Empidoidea.
DISCUSSION
Presence or absence of metacephalic rod
The larval characters discussed in this paper are
rearranged below.
(1) Head strongly sclerotized and not com-
pletely retractile within thorax: Stratiomyoidea,
Xylophagoidea, Therevidae and Scenopinidae;
plesiomorphic in relation to the Tabanoidea, Ver-
mileonidae, Nemestrinoidea, Empidoidea and
many Asiloidea.
(2) Head completely retractile within thorax:
Tabanoidea, Vermileonidae, Nemestrinoidea,
Asiloidea (except for Therevidae and Scenopini-
dae) and Empidoidea; apomorphic in relation to
Nematocera, Stratiomyoidea, Xylophagoidea and
some Asiloidea.
(3) Head with paired metacephalic rods:
Xylophagoidea (except for the Pantophthalmi-
dae), Empidoidea and some Asilidae.
(4) Head with a single median metacephalic
rod: Therevidae, Scenopinidae, Mydidae,
Apioceridae and most Asilidae.
(5) Head without metacephalic rod: Stra-
tiomyoidea, Pantophthalmidae, Tabanoidea, Ver-
mileonidae, Nemestrinoidea and Bombyliidae.
(6) Posterior spiracles closer together on the
last abdominal segment: Stratiomyoidea, Pantoph-
thalmidae, Xylophagoidea, Tabanoidea, Ver-
mileonidae, Nemestrinoidea and Empidoidea; ple-
siomorphic in relation to the Asiloidea.
(7) Posterior spiracles more widely separated
from each other on the apparent penultimate
abdominal segment: Asilidae, Mydidae, Apiocer-
idae and most Bomyliidae; apomorphic in relation
to other orthorrhaphous Brachycera (except for
Therevidae and Scenopinidae).
(8) Posterior spiracles more widely separated
from each other on antepenultimate abdominal
segment: Therevidae and Scenopinidae; apomor-
phic in relation to other Asiloidea.
It remains doubtful to me whether the presence
of paired metacephalic rods in the Xylophagidae s.
lat. is apomorphic or plesiomorphic in relation to
the Stratiomyoidea, Pantophthalmidae, Taba-
noidea, Vermileonidae, Nemestrinoidea and
Bomyliidae. The metacephalic rod is absent in the
Nematocera but present in the Xylophagidae s.
lat., Asiloidea (except for Bombyliidae), and
Empidoidea. It seems therefore that the ancestor
of the orthrrhaphous Brachycera had the
metacephalic rod.
When apomorphic, presence of the metacephal-
ic rod evolved independently in the Asiloidea
854 A. NAGATOMI
(except for Bombyliidae) and Empidoidea respec-
tively. When plesiomorphic, loss of the
metacephalic rod occurred independently in the
Stratiomyoidea+ Pantophthalmidae, Vermileoni-
dae, Tabanoidea+Nemestrinoidea, and Bomby-
liidae respectively. Either of these two cases is
equally conceivable.
Krivosheina [3] suggested monophyly of the
Stratiomyoidea + Pantophthalmidae + Tabanoidea
+Nemestrinoidea+Bombyliidae or that of the
Xylophagoidea-+ Asiloidea (except for Bomby-
liidae) according to the absence or presence of the
metacephalic rod. The above hypothesis is not
deniable, but the various factors discussed in this
paper weaken the argument for it in the present
state of our knowledge.
Apomorhy or plesiomorphy in some characters of
male genitalia
Nagatomi ({11] p. 154) wrote, “(1) The tergum 9
is strongly arched in the genera Rachicerus,
Xylophagus, Exeretonevra, and Pelecorhynchus
but rather flat in Pantophthalmus, Coenomyia et
al., Heterostomus, Rhagio et al. etc., and among
the former four genera (2) the postero-lateral part
of tergum 9 is developed ventro-inwardly into a
plate or flap in Rachicerus and Pelecorhynchus but
not in Xylophagus and Exeretonevra. It is ques-
tionable whether these two characters are ple-
siomorphic or are developed in each group of the
lower Brachycera independently of the phylogene-
tic relationship. The former view seems to be
more probable.” In the last sentence quoted
above, read “latter” for “former”.
The arched tergum 9 and well-developed surstyli
are beyond doubt apomorphic characters. These
two characters are distributed in the Xylophagidae
s. lat. and the Pelecorhynchidae, and their inde-
pendent development was considered to be less
probable in Nagatomi [11]. The genera Rachi-
cerus, Exeretonevra and Pelecorhynchus are each
markedly specialized in some external characters
as already discussed in this paper.
It is better to refrain from further comments on
the various structures of the male genitalia with
regard to the apomorhy or plesiomorphy, because
of the difficulty of assessment.
Two types in primitive creatures
A number of archaic creatures must specialize to
a certain or extreme degree in order to survive
long, while not a few ancient living things survive
without strong specialization. Thus, there are two
forms in the Xylophagidae s. lat. and Rhagionidae
s. lat. which constitute the stem or base of the
phylogenetic tree in the orthorrhaphous Brachy-
cera.
Specialized form: Rachiceridae (the antennal
flagellum is markedly specialized); Exeretonevri-
dae (wing venation); Pelecorhynchus of Pele-
corhynchidae (cheek, palpus, and 2nd submarginal
cell).
Generalized form: Coenomyiidae; Heterostomi-
dae; Glutops and Pseudoerinna of Pelecorhynchi-
dae; Rhagionidae.
As a matter of course, to find a synapomorphic
character is very difficult in the generalized form.
Phenetic taxonomy and cladistic taxonomy
The Nematocera and the orthorrhaphous
Brachycera each seems to be not monophyletic but
paraphyletic, and many higher taxa of the insects
may remain as paraphyletic ones.
When the principle of cladistic taxonomy is
applied strictly, some taxa can become unnatural
instead.
For instance the sister group of the snakes and
lizards is the crocodiles+birds, and that of the
orang-utan is gorilla+chimpanzee+man. The
oldest ancestor of the birds would greatly resemble
the crocodiles.
Even if the monphyly of the Vermileonidae +
Apsilocephalidae + Empidoidea + Cyclorrhapha
was recognized as true, the Empidoidea et al.
would still be placed in the orthorrhaphous
Brachycera, because the Cyclorrhapha has devi-
ated so far from the Empidoidea etc.
Validity of small families
There are many small families, namely,
Rachiceridae, Xylophagidae, Coenomyiidae,
Heterostomidae, Exeretonevridae and Pele-
corhynchidae, of which the first five belong to the
Xylophagidae s. lat. and the last to the Rhagioni-
dae s. lat. All of them could be reduced to
Phylogeny of Orthorrhaphous Brachycera 855
subfamily status and in that case, Pelecorhynchi-
nae and Glutopinae (or plus Pseudoerinniinae) of
the family Rhagionidae may be recognized. For
explanatory convenience, family status is given to
the taxa above in this paper. In any case, the
choice between “family” and “subfamily” will be
determined by personal preference for the taxa
above.
On the other hand, the Vermileonidae and
Apsilocephalidae should each be treated as an
independent family, because they are so isolated
from each other and from other taxa of the orthor-
thaphous Brachycera.
The validity of the Hilarimorphidae as an inde-
pendent family is left for the future.
ADDITIONS TO NAGATOMI [25]
Some notes are added to Nagatomi [25] on the
history of some families of Deptera, chiefly those
of the lower Brachycera.
Canthyloscelidae
The genera Canthyloscelis and Hyperoscelis
(Canthyloscelidae) and Synneuron (Synneuridae)
have the following common characters: “head cap-
sule greatly reduced and membranous, with
mouthparts unsclerotized and indistinct” (Wood
and Borkent [42]).
Wood and Borkent ([42] p. 1354) wrote, “Hut-
son [43] contended that the Scatopsidae are de-
rived from Synneuron as a terminal branch of one
of four lineages making up the Synneuridae plus
Canthyloscelidae. If his contention is so, the head
capusule of the larva must have been lost at least
twice (thrice if the larva of Exiliscelis also lacks a
fully formed head capsule), or else a fully formed
head capsule must have been reacquired in the
Scatopsidae. We consider both of these possibili-
ties unlikely and have abandoned Hutson’s clado-
gram (his fig. 26) in favor of a sister-group rela-
tionship between the Scatopsidae and the Syn-
neuridae [= Canthyloscelis + Hyperoscelis + Exilis-
celis + Synneuron].”
Pelecorhynchidae
Daniels [44] described 2 new species of Pele-
corhynchus from Victoria and Western Australia
(see also Daniels [45]). These 2 species were
accidentally omitted in Nagatomi [46, 25].
Nagatomi ((25] p. 14) wrote, “The genus Glu-
tops was placed in the Pelecorhynchidae by several
North American workers. If this treatment is
correct, Glutops would be a more plesiomorphic
northern-hemisphere sister-group of Pelecorhyn-
chus. However, the phylogenetic position of Glu-
tops is not necessarily established and will be
discussed in a separate paper.”
In the present instance, I have come to believe
that Glutops and Pseudoerinna (= Bequaertomyia)
are more closely related phylogenetically to Pele-
corhynchus than to other genera of Rhagionidae s.
lat., following Woodley and Teskey [18, 23].
Presence or absence of multiple species groups
within one genus, each of which stretches over
Australia and South America
On p. 29 in Nagatomi [25], add the figure 3 in
Hennig [47].
CONCLUDING REMARKS
(1) The Xylophagidae s. lat. seems to be older
in Origin than the Rhagionidae s. lat.
(2) The systematic position of various taxa is
judged as follows: Pantophthalmidae: either Stra-
tiomyoidea or Xylophagoidea; Exeretonevra:
Xylophagidae s. lat.; Austroleptis: Rhagionidae or
close relative of Rhagionidae; Glutops: Pele-
corhynchidae; Vermileonidae: sister group of Apsil-
ocephalidae + Empidoidea+Cyclorrhapha; Nem-
estrinoidea: sister group of Tabanoidea; Bombyl-
iidae: Asiloidea.
(3) There are some definite autapomorphic or
synapomorphic characters for Coenomyiidae,
Pelecorhynchidae + Rhagionidae, and Athericidae
+ Tabanidae, respectively.
(4) There are some possible synapomorphic
characters for Rachiceridae+ Xylophagidae, Ex-
eretonevridae + Heterostomidae, and Vermileoni-
dae + Apsilocephalidae + Empidoidea+ Cyclor-
rhapha, respectively.
(5) There is one case of circumstantial evi-
dence for monophyly of Stratiomyoidea+
Xylophagoidea.
(6) Various problems are discussed as follows:
856 A. NAGATOMI
presence or absence of metacephalic rod; apomor-
phy or plesiomorophy in some characters of male
genitalia; two types in primitive creatures; phene-
tic taxonomy and cladistic texonomy; validity of
small families in the orthorrhaphous Brachycera.
(7) Some notes are added to Nagatomi [25].
(8) Figures 1-3 show the most plausible phy-
logenetic relationships of the orthorrhaphous
Brachycera.
ACKNOWLEDGMENTS
This paper is dedicated to Dr. Kintaro Baba (in
Niigata Pref.) for his interest, encouragement and help
which greatly facilitated its preparation.
My heart-felt thanks are also expressed to Dr. Leif
Lyneborg (Zoological Museum, Copenhagen), Professor
Toyohei Saigusa (Kyushu University, Fukuoka), Mr. K.
G. V. Smith (formerly British Museum [Natural His-
tory], London), Dr. Brian R. Stuckenberg (Natal
Museum, Pietermaritzburg, South Africa) and Dr. H. J.
Teskey (formerly Biosystematic Research Institute,
Agriculture Canada, Ottawa) for their long-term help in
many ways. Mr. Smith has also kindly checked the
English of this manuscript.
I have learned much from the unpublished manuscript
on larval stages of primitive Bombyliidae by Dr. D. K.
Yeates (American Museum of Natural History, New
York) and Dr. M. E. Irwin (University of Illinois,
Champaign) to whom I am much indebted. A part of this
paper was read at the 19th International Congress of
Entomology (Beijing, China) under the chairmanship of
Dr. G. C. D. Griffiths (University of Alberta, Edmon-
ton) to whom I am also deeply indebted.
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ZOOLOGICAL SCIENCE 9: 859-874 (1992)
© 1992 Zoological Society of Japan
Misophriopsis okinawensis sp. nov. (Crustacea: Copepoda)
from Hyperbenthic Waters off Okinawa, South Japan,
with Definitions of Related Genera Misophria Boeck,
1864 and Stygomisophria gen. nov.
Susumu Ontsuka!, Rony Huys’, GEorrrEY A. BoxsHALl”
and Tatsunort It6°
‘Fisheries Laboratory, Hiroshima University, Takehara, Hiroshima 725, Japan,
"Department of Zoology, The Natural History Museum, Cromwell Road,
London SW7 5BD, England, and 3The Seto Marine Biological
Laboratory, Kyoto University, Wakayama 649-22, Japan
ABSTRACT—A new misophrioid copepod Misophriopsis okinawensis (Crustacea) is reported from
Kume Island, Okinawa, South Japan. A full description of the new misophrioid is presented, together
with supplementary notes on the males of Misophria pallida Boeck, 1864.
Consideration of the
phylogeny of the new species led to a reassessment of the affinities between all the named species of
Misophria Boeck, 1864 and Misophriopsis Boxshall, 1983. A new genus, Stygomisophira, is recognised
based on Misophria kororiensis Boxshall et Iliffe, 1987. Diagnoses are presented of all three genera.
INTRODUCTION
During taxonomic and phylogenetic studies of
the marine hyperbenthic copepod fauna of Japan
by one of the authors (SO) (e.g., [1, 2]), a new
species of Misophriopsis Boxshall, 1983 (Copepo-
da: Misophrioida) was collected at a depth of ca.
170 m off Kume Island, Okinawa, South Japan.
The new species is described in detail in the
present paper. The misophrioid copepods were
infested by a new small crustacean ectoparasite,
whose description is given in another paper by the
same authors as the first record of the subclass
Tantulocarida in the North Pacific region (see [3]).
The genus Misophriopsis comprised two sepcies,
namely, the type speices, M. dichotoma Boxshall,
1983 [4] and an undescribed new species referred
to as Misophriopsis sp. nov. by Boxshall [5] and by
Huys and Boxshall [6]. The original diagnosis of
Misophriopsis emphasized the presence of a dis-
crete 1-segmented endopod on the female fifth leg
Accepted May 28, 1992
Received January 31, 1992
" To whom all correspondence should be addressed.
3 Deceased on April 8th, 1990
(see [4]) but the discovery of a similar discrete
endopod in female Misophria pallida Boeck, 1864
by Huys and Boxshall [6] makes a reexamination
of the validity by these two genera necessary. The
group of species currently placed in Misophria
comprises the type species, M. pallida, plus two
additional species, M. kororiensis Boxshall and
Iliffe, 1987 [7] and M. sinensis Boxshall, 1990 [8].
Distinct genera can be recognized within this com-
plex of species by reference, in particular, to the
segmentation of the antennules, the form of the
seminal receptacle of the female, and the structure
and armature of the fifth and sixth legs of both
sexes. Based on these characters, new differential
diagnoses of Misophriopsis, Misophria, and a new
genus accommodating M. kororiensis are pre-
sented here. The male of Misophria pallida
Boeck, 1864 is also redescribed in order to redefine
these three genera.
One of the authors, Tatsunori Ito deceased
suddenly on April 8, 1990 before he finished
describing the new host misophrioid copepod and
the new parasitic tantulocaridan. This study is
dedicated to the late Dr. T. It6 by the first three
authors (SO, RH, GAB), in honor of his excellent
860 S. OntsuKA, R. Huys et al.
taxonomic and phylogenetic works on maxillopo-
dans.
MATERIALS AND METHODS
The new misophrioid copepods were collected
with an originally designed sledge-net (mouth
area: 1450 mm X 326 mm; mesh size: 0.33 mm) at a
depth of about 167 m off Kume Island, Okinawa,
South Japan (26°17.9'N, 126°54.2’E) on 23 May
1989 (local time: 0824-0858). The sledge-net was
towed along the bottom at a speed of 2 knots by
the T/RV Toyoshio-maru of Hiroshima University
for ca. 5 minutes while the depth was measured by
a remote-sensing system (Furuno-denki, Color net
recorder CN-8). The specimens were fixed in 10%
neutralized formalin/sea-water immediately after
collection. The new misophrioids were examined
with differential interference microscopes (Leitz
Dialux 20; Nikon Optiphot) and a scanning elec-
tron microscope (JEOL JST-T20). All types of the
new species of Misophrioida are deposited in the
collections of The Natural History Museum,
London.
Misophria pallida was redescribed based on
three adult males collected from deep sediment of
an unspecified locality in Norway (Zoologisk
Museum, Oslo (G. O. Sars collection), reg. no.
F20793).
Redefinition of Misophriopsis Boxshall,
1983 and description of Misophriopsis
okinawensis sp. nov.
Family Misophriidae Brady
Genus Misophriopsis Boxshall, 1983
Diagnosis. Female antennule 18-segmented;
male antennule 12-segmented, with aesthetasc on
segment I. Seminal receptacle produced trans-
versely. Fifth legs without intercoxal sclerite in
both sexes; female 5th leg with undivided pro-
topod, endopod a small unisetose lobe, free or
fused to protopod, proximal exopod segment un-
armed, distal exopod segment with 1 seta on each
side of apical spine; male Sth leg with undivided
protopod, unisetose free endopod and_ 3-
segmented exopod, lst exopod segment unarmed,
2nd with 1 inner seta, 3rd with 1 inner seta and 1
seta on each side of apical spine; 6th legs with 1
long seta and 2 short spines in female, with 1 spine
and 2 setae in male.
Type species.
shall, 1983.
Remarks. Remarks on the genus Misophriop-
sis is presented after the description of the new
species described below.
Misophriopsis dichotoma Box-
Misophriopsis okinawensis sp. nov.
(Figs. 1-9)
Types. All types collected from near sandy
bottom (ca. 167m in depth) off Kume Island,
Okinawa, Japan, and deposited in The Natural
History Museum, London. Holotype: adult
female, dissected and mounted on glass slides, BM
(NH) 1991. 151. Paratypes: two adult females and
two adult males, dissected and mounted on glass
slides, BM (NH) 1991. 152-155; 25 adult females
and two adult males, whole specimens, BM (NH)
1991. 156-182; 15 adult females, whole specimens,
BH (NH) 1991. 376-390.
Body length. Female: mean+standard devia-
tion=0.73+0.03 mm (range =0.66—0.79 mm,
number examined=28). Male: 0.54+0.01 mm (r
=0.52-0.55 mm, n=5).
Description. Female (holotype: Figs. 1-A~F,
6-A~D; paratype: Figs. 2~5). Body (Fig. 1-A)
compact, 0.78mm in length; prosome oval in
dorsal view, about 3 times longer than urosome
(Fig. 1-A, B). First pedigerous somite entirely
concealed beneath carapace-like expansion from
posterior end of maxilliped-bearing somite. Third
and 4th pedigerous somites produced posteriorly.
Urosome (Figs. 1-B, 2-C) 5-segmented; 5th
pedigerous somite posterolaterally produced into
acute process on both sides; genital and 1st abdo-
minal somites fused to form genital double-somite;
original subdivision marked by difference in width
between anterior and posterior parts of double-
somite; pair of minute copulatory pores located in
a small transverse median slit on mid-ventral sur-
face of double-somite, each of which leading to a
common, transversely produced seminal recepta-
cle; pair of slit-like genital pores (Figs. 2-C, 3-C)
located ventrolaterally on double-somite and co-
New Misophrioid Copepod from Okinawa 861
0.1mm
C-E
Fic. 1. Misophriopsis okinawensis sp. nov. Female (holotype). A. Habitus, dorsal view; B. Urosome, dorsal view;
C. Antennule; D. Antenna; E. Basal two exopod segments of antenna; F. Distal exopod segment of antenna.
Male (paratype). G. Habitus, dorsal view.
vered by operculum derived from leg 6, armature
consisting of 2 spinous processes and 1 seta; 4th
somite completely concealed beneath 3rd; anal
somite fringed with minute prominences dorsopos-
teriorly. Caudal ramus (Figs. 3-D, 4-A) wider
than long, bearing 1 dorsal (VII in Fig. 4-A), 1
anterolateral accessory (I), 1 anterolateral (II), 1
posterolateral (III), 1 terminal accessory (VI) and
862 S. Outsuka, R. Huys et al.
Fic. 2. Misophriopsis okinawensis sp. nov. Female (paratype). A. Ventral view of oral area and rostrum; B. Lateral
view of oral area and rostrum; C. Legs 5 and 6 and genital double-somite, dotted area indicates seminal
receptacle. A,: Antennule; R.: Rostrum; L.: Labrum; P.: Paragnath; M.: Mandible; IMS.: Intermaxillary
swelling.
2 stout, terminal spinulose setae (IV and V). _ pair of sensilla near its pointed tip. Naupliar eye
Rostrum (Fig. 2-A, B) partly fused with labrum, absent. Labrum (Fig. 2A, B) covered with numer-
posteroventrally produced, and furnished with a ous minute spinules, bearing row of fine hairs near
863
New Misophrioid Copepod from Okinawa
——
{ Pith,
4 Hl pS
ANY We x
aa ————
LE
Fic. 3. Misophriopsis okinawensis sp. nov. Female (paratype). A. Mandibular gnathobase; B. Mandibular palp; C
Leg 6 and genital and copulatory pores on genital double-somite, lateral view; D. Caudal ramus, ventral view.
its posterior margin and pair of short prominences
on both sides of posterior end. Pair of paragnaths
(Fig. 2-A, B) located posterior to end of labrum,
ornamented with fine spinules on tip; intermaxil-
lary swelling (Fig. 2-A, B) arising from mid-
ventral surface, with row of minute spinules along
posterior margin.
Antennule (Fig. 1-C) 18-segmented. Segmental
distribution of armature elements compiled in
Table 1. Antenna (Fig. 1-D~F): coxa bearing row
864 S. OntsuKA, R. Huys et al.
yA
SEs >
5 DS
PASS
Zl
7,
LT,
Fic. 4.
of fine setules near anterior margin; basis with 2
apical setae of unequal lengths and patch of fine
spinules on posterior surface. Endopod 3-
segmented; segment 1 bearing 2 small, subdistal
inner setae of unequal lengths and patch of minute
Misophriopsis okinawensis sp. nov. Female (paratype). A. Caudal ramus, lateral view; B. Maxillule.
spinules on posterior surface; segment 2 with 2
lateral and 3 distal setae along inner margin;
segment 3 bearing 6 long setae and short seta
apically. Exopod 6-segmented; setal formula as
follows: 0, 2, 1, 1, 1, 3. Mandibular gnathobase
New Misophrioid Copepod from Okinawa 865
OO UE Te
BAG WG Z
ul y CE Cas
ad a ieee
SSS
lle
Lp
LF
4 Lz of
<< SI r
Fic. 5. Misophriopsis okinawensis sp. nov. Female (paratype). A. Maxilla, setae of allobasis and endpod omitted;
B. Maxillary allobasis and endopod; C. Maxilliped; D. Distal segment of maxillipedal endopod.
(Fig. 3-A) with 5 multicusped teeth, 3 serrate
blades and 1 spiniform seta; ornamentation of fine
spinules and relatively long setules present on
anterior surface near palp. Mandibular palp (Fig.
3-B) biramous; basis bearing patches of fine spi-
nules and inner medial seta. Endopod 2-
segmented; proximal segment with inner subter-
minal seta; distal segment bearing 1 medium
866 S. OutsukA, R. Huys et al.
SSS
SK
Fic. 6. Misophriopsis okinawensis sp. nov. Female (holotype).
surface; C. Leg 3, anterior surface; D. Leg 4, anterior surface.
length, 1 short and 6 long setae. Exopod 5-
segmented; setal formula as follows: 1, 1, 1, 1, 2.
Maxillule (Fig. 4-B): praecoxal arthrite with 7
strong spines and 8 setae, 2 of which arising from
anterior surface; coxal endite bearing 1 thick se-
trate seta and 5 pinnate setae distally; proximal
basal endite with 4 apical setae, distal basal endite
armed with 4 setae; epipodite of coxa with 7 setae
of unequal lengths. Endopod comprising single
compound segment, representing fused Ist to 3rd
A. Leg 1, anterior surface; B. Leg 2, anterior
segments; armature divided into groups of 3 inner
medial, 3 inner subterminal and 6 terminal setae
representing original segmental elements. Exopod
1-segmented, bearing 3 inner lateral and 5 terminal
setae and relatively long setules along inner and
outer margins. Maxilla (Fig. 5-A, B): praecoxa
partly fused with coxa, with 2 endites, proximal
armed with 6 setae, distal with 3 setae; coxa with 2
endites and outer patch of fine spinules, each
endite with 3 distal setae; allobasis (Fig. 5-B)
New Misophrioid Copepod from Okinawa 867
Fic. 7. Misophriopsis okinawensis sp. nov. Male (paratype). A. Antennule; B. Antennulary segments 3 to 7, dorsal
view; C. Antennulary segment 3 to 7, ventral view; D. Leg 5, anterior surface; E. Leg 6, anterior surface.
derived from fusion of basis and 1st endopodal
segment; produced into strong curved claw and
bearing 6 setae (3 of which are derived from 1st
endopodal segment); free endopod indistictly 3-
segmented, with setal formula 2, 2, 4. Maxilliped
(Fig. 5-C, D) having 4 endites on syncoxa with
setal formula 0, 1, 3, 2; row of long spinules along
outer medial margin; proximal patch of minute
spinules and irregular rows of small spinules near
bases of setae on middle and subdistal endites;
868 S. OntsuKA, R. Huys et al.
oad
Fic. 8.
Misophriopsis okinawensis sp. nov. SEM micrographs of female. A. Prosome, dorsal view; B. Urosome,
dorsal view; C. Cephalosome, lateral view; D. Epicuticular ornamentation on cephalosome. Scale bars=100 «m
(A, C); 50 um (B, D).
basis with 3 spinulose inner setae medially and row
of long spinules proximally, fringed with short
spinules along posterior half of inner margin; free
endopod 5-segmented; setal formula: 2, 2, 2, 2, 5.
The armature formula of legs 1 to 4 is shown in
Table 2 and compared with two other congeneric
species, Misophriopsis dichotoma and M. sinensis
(Boxshall, 1990) comb. nov. Leg 1 (Fig. 6-A):
coxa with relatively long spinules near base of
plumose inner distal seta; intercoxal sclerite with
spinulose row along distal margin; basis bearing
row of spinules on anterior surface, and its medial
New Misophrioid Copepod from Okinawa 869
- Z TSS
Fic. 9. Misophriopsis okinawensis sp. nov. SEM micrographs of female. A. Labrum, pore indicated by an arrow; B.
Leg 6. Scale bars=50 um (A); 10 um (B).
distal margin expanded into lamellar plate; inner
distal spine on basis reaching beyond distal angle
of 1st endopod segment; outer distal angle of 2nd
endopod segment bifid; outer margins of Ist and
2nd exopod segments lamellar, fringed with mi-
nute serrations. Leg 2 (Fig. 6-B): inner distal
margin of basis produced into acute tip; its medial
distal end expanding into fan-like structure with
serrate inner margin; inner distal angle of 2nd
endopod segment irregularly produced; inner dis-
tal angles of Ist and 2nd exopod segments pro-
duced into blunt process. Leg 3 (Fig. 6-C) similar
to leg 2, but basis bearing outer medial seta instead
of spine and 3rd exopod segment with 5 inner
setae. Leg 4 (Fig. 6-D) covered with numerous
spinules, in particular, on posterior surface; inner
distal end of basis not produced as in legs 2 and 3;
intercoxal sclerite relatively small compared with
in preceding legs. Leg 5 (Fig. 2-C) indistinctly
biramous; protopod comprising fused coxa and
basis; inner distal seta on inner process represent-
ing endopod partly separated from basis by pro-
ximal constriction; outer basal seta present. Pro-
topod fringed by minute spinules along inner mar-
gin. Exopod 2-segmented; proximal exopod seg-
ment unarmed; distal segment laterally and
medially bearing fine spinules, and distally serrate
spine, inner small and outer medium-length setae.
Leg 6 (Figs. 2-C, 3-C) represented by genital oper-
culum bearing outer seta on low cylindrical process
and 2 small blunt processes.
Male (paratypes). Body (Fig. 1-F) 0.54 mm in
length, much smaller than in female. Urosome
6-segmented; Sth pedigerous somite with acute
posterolateral angles; genital somite large; 3rd
abdominal somite small, largely concealed beneath
2nd. Both (Fig. 7-A~C) 12-
segmented; geniculate with geniculation between
10th (XIX-XX) and 11th (XXI-XXII]) segments;
fusion pattern and distribution of armature ele-
ments shown in Table 1. Leg 5 (Fig. 7-D) bira-
mous; compound protopodal segment fringed by
minute spinules along inner margin, and bearing 1
seta derived from basis; endopod represented by
small subcircular segment, bearing small seta dis-
tally. Exopod 3-segmented; proximal segment
antennules
870 S. OuntsuKA, R. Huys et al.
TABLE 1.
pallida.
numeral.
Armatures of antennules of Misophriopsis okinawensis sp. nov. and Misophria
Number of antennulary segment of ancestral copepod represented by Roman
Number of setae on the segment represented by Arabic numeral: a+b=
(number of anterior setae)+(number of posterior setae).
ae: Aesthetasc; proc:
Spinous process derived from modification of setal element
M. okinawensis (female)
I 1
II-VII 12
VIII 2
IX-XII 8
XIll 2
XIV 2
XV 2
XVI 2+ae
XVII 2
XVIII 2+ae
XIX D
xX 2
XXI 2+ae
XXII 1
XXIII 1
XXIV 1+1
XXV 1+1-+ae
XXVI-XXVIII 7+ae
M. okinawensis (male)
I 1+ae
II-VI 9+2ae
VII 2+ae
VIII 2
IX-XIII 10+ 2ae
XIV 2+ae
XV-XVI 4+ae
XVII 2
XVIII 2+ae
XIX-XX 2+ proc
XXI-XXIII 3+ ae
XXIV-XXVIII 11+2ae
unarmed; middle segment with thick, plumose seta
at distal inner angle; apical segment with 1 inner
seta and 1 seta on each side of apical serrate spine.
Leg 6 (Fig. 7-E) forming opercular plate overlying
genital opening, bearing large serrate inner spine
and 2 plumose setae.
Body surface observed with SEM. The body
surface of Misophriopsis okinawensis is almost
M. pallida (female)
I 1
II-VI 14
IX-XII 8
XI 2
XIV 2
XV 2
XVI 2+ae
XVII 2
XVIII 2+ae
XIX 2
XX 2
XXI 2+ ae
XXII 1
XXIll 1
XXIV 1+1
XXV 1+1+ae
XXVI-XXVIII 7+ae
M. pallida (male)
I 1
II-VI 9+2ae
Vil 2+ ae
VII 2
IX-XIII 10+ ae
XIV 2+ae
XV 2
XVI 2+ae
XVII 2
XVII 2+ae
XIX-XX 2+ 2proc
XXI-XXIIl 3-+ae+ proc
XXIV-XXVIII 11+2ae
entirely covered with shallow, epicuticular lamel-
lae except for the anterior half of the dorsal
cephalic shield which has a smooth surface with a
number of hair-like sensilla and small pores but no
lamellae (Fig. 8-A, C, D). The lamellae are per-
pendicular to the surface on the prosome (Fig.
8-D), whereas they incline posteriorly on the uro-
some (Fig. 8-B). Cone organs lacking on the
New Misophrioid Copepod from Okinawa 871
TaBLeE2. Seta and spine formula of legs 1 to 4 of Misophriopsis okinawensis sp. nov., M. dichotoma and M.
sinensis comb. nov. Seta represented by Arabic numeral, and spine by Roman numeral. o=outer
border of segment; t=terminal border of segment; i=inner border of segment
Coxa Basis Endopod Exopod
1 2 3 1 2 3
(0) i (0) 1 ©) i (0) i (0) 1 (0) i (0) 1 0 t i
Misophriopsis okinawensis sp. nov.
Leg 1 (Ds A wile Th, Mul Gg mam ied anh 2
Leg 2 ge tile) anil Mae Veh fade Oa
Leg 3 CRemelane i MOCO” yt Poimeks
Leg 4 (Eee eee TONe Os teen !2
M. dichotoma’
Loo 1) CRN i ce es ee
Leg 2 0 1 1 0 0 1 0 2
M. sinensis comb. nov.”
Leg 1 0 1 1 I @) 1 0 2
Leg 2 0 1 1 0 0 1 0 2
1 2 3 I 1 I Ill It 3
2 3 I 1 I 1 Ill I 4
1 2 3 I 1 I 1 Ill I 5
1 2 2 I 1 I 1 Ill I 5
1 2 3 I 1 I 1 Ill I
1 2 3 I 1 I 1 Ill I 5
1 2 3 I 1 I 1 Ill I 4
1 2 3 I 1 I 1 Ill I 5
" Armature formula of legs 3 and 4 the same as in M. okinawensis but the third endopod segments of legs 3 and 4
missing.
2 Armature formula of legs 3 and 4 the same as in M. okinawensis.
lateral side of cephalosome (Fig. 8-C) as in Benth-
omisophria palliata Sars, 1909 and Misophriopsis
dichotoma Boxshall, 1983 [4, 6]. The urosome is
covered with lamellae except for the anal somite,
as in other misophrioids (Fig. 8-B). The fifth
pedigerous somite has a relatively large pore on
the dorsomedial surface; the genital somite, anter-
ior and posterior dorsomedial pores; the 1st abdo-
minal somite, posterior medial pore dorsally; the
fourth somite, anterior pore dorsomedially.
The labrum is densely covered with ornamenta-
tion consisting of dentate scales separated by open
smooth areas (Fig. 9-A). Fig. 9-A shows that the
rostrum and the labrum are partly separate from
each other. A pore located medially on the
anterior part of labrum is arrowed in Fig. 9-A. Leg
6 of the female (Fig. 9-B) is armed with a plumose
seta on a low cylindrical process, a small spine, and
smooth and serrate processes.
Etymology. The specific name “okinawensis”
is derived from the type locality, Okinawa Prefec-
ture.
Remarks. Misophria sinensis from Hong Kong
is known only from the female but conforms to this
new generic diagnosis of Misophriopsis in the
18-segmented antennule and the absence of any
armature from the proximal exopod segment of
the fifth legs. It is here transferred to Misophriop-
sis. The nature of the seminal receptacle of M.
sinensis could not be confirmed in the only avail-
able specimen. The genus Misophriopsis now
comprises the type species plus M. sinensis (Box-
shall, 1990) comb. nov. and M. okinawensis sp.
nov. These species differ in the armature of legs 1
and 2 (see Table 2): M. okinawensis and M. dicho-
toma have a spine on the outer border of the basis
while M. sinensis possesses a seta in this position;
the numbers of setae along the inner margin of the
third exopod segments of legs 1 and 2 are more
reduced in M. okinawensis than in M. dichotoma
and M. sinensis.
The undescribed Misophriopsis species from the
North Atlantic off the coast of Norway, mentioned
by Boxshall [5] and illustrated in part by Huys and
Boxshall [6] differs significantly from the new
concept of Misophriopsis. Its systematic position
will be considered in a separate account after it has
been fully described. Misophria kororiensis, de-
scribed from a subtidal anchialine cave in Palau
[7], cannot be placed in either Misophria or Mis-
872 S. Outsuka, R. Huys et al.
ophriopsis. Accordingly a new genus is proposed
to accommodate M. kororiensis. The differential
diagnosis of the new genus is given below.
Redefinition of Misophria Boeck, 1864 and
establishment of Stygomisophria gen. nov.,
with description of male Misophria
pallida Boeck, 1864
Genus Misophria Boeck, 1864
Diagnosis. Female antennule 17-segmented;
male antennule 13-segmented, without aesthetasc
on segment I. Seminal receptacle not transversely
produced. Fifth legs without intercoxal sclerite in
both sexes; female 5th leg with undivided pro-
topod, endopod a small bisetose free lobe, proxim-
al exopod segment with outer seta, distal exopod
segment with 1 seta on each side of apical spine;
male Sth leg with bisetose endopod fused to undi-
vided protopod, exopod as in female; 6th legs
armed with 1 long seta and 2 short spines in
female, with 2 setae in male.
Type species. Misophria pallida Boeck, 1864,
monotypy.
Remarks. The genus Misophria is disting-
uished from the genus Misophriopsis in: (1) the
numbers of antennulary segments of both sexes;
(2) the presence or absence of an aesthetasc on
antennulary segment I; (3) the shape of seminal
receptacle; (4) the armature elements of legs 5 and
6 of both sexes.
Misophria pallida Boeck, 1864
(Fig. 10)
Both sexes of Misophria pallida were described
by Sars [9] but the description contains insufficient
detail for a full comparison to be made with the
other known species currently attributed to Mis-
ophria (see [7, 8]). Huys and Boxshall [6] illus-
trated some features of M. pallida, including the
female antennule, the rami of the mandibular
palp, the female fifth leg and the female genital
region including the sixth legs. Supplementary
descriptions of the antennules and the fifth and
sixth legs of the males are presented below in order
to compare the male of M. pallida with that of
Misophriopsis okinawensis sp. nov.
Male antennule (Fig. 10-A) 13-segmented, seg-
mental fusion pattern and setation pattern summa-
rized in Table 1; compound segments II-VI and
IX-XIII without any incomplete sutures marking
fusion planes. Segments XV and XVI partly fused
along posterior surface; sheath present on segment
XV (arrowed in Fig. 10-B) but with anteroventral
margin of sheath integrated into segment leaving
shallow transverse furrow (arrowed in Fig. 10-C).
Geniculation in neocopepodan position between
compound segments XIX-XX and XXI-XXIII.
Male 5th legs (Fig. 10-D) separate, intercoxal
sclerite lacking; leg comprising compound pro-
topodal segment bearing outer basal seta and 2
setae on inner distal margin representing endopod
armature; endopod completely incorporated into
compound protopodal segment, with no vestige of
suture remaining; exopod 2-segmented; proximal
segment with seta on outer margin, distal segment
produced into spinous process at outer distal
angle, distal margin armed with long outer seta,
bilaterally serrate spine and shorter, naked, inner
angle seta (Fig. 10-D). All segments with surface
and marginal ornamentation of fine spinules.
Male 6th leg (Fig. 10-E): a transverse plate
closing off genital aperture on each side; armed
with 2 setae and ornamented with spinular row
along free margin.
Stygomisophria gen. nov.
Diagnosis. Female antennule 18-segmented.
Seminal receptacle not produced transversely.
Female 5th leg with intercoxal sclerite and sepa-
rate coxa and basis, endopod represented by | seta
on inner distal margin of basis, proximal exopod
segment with outer seta, distal exopod segment
with 1 seta on each side of apical spine; female 6th
leg with 1 long seta and 1 small spine. Male
unknown.
Type species. Misophria kororiensis Boxshall
et Iliffe, 1987, monotypy.
The new genus is distinguished from
both Misophria and Misophriopsis by the retention
of an intercoxal sclerite and separate coxa and
basis in the female fifth leg. It also differs from
Misophria in segmentation of the female antennule
Remarks.
873
New Misophrioid Copepod from Okinawa
0.05 mm
A
o.05mm B,C
Fic. 10. Misophria pallida Boeck, 1864. Male. A. Rostrum and antennule, geniculation indicated by an arrowhead;
B. Antennulary segments 2 to 8, dorsal surface, distal margin of sheath on segment 7 indicated by arrowheads,
aesthetascs omitted; C. Antennulary segments 2 to 8, ventral surface, distal margin of sheath on segment 7
indicated by arrowheads; D. Leg 5, anterior surface; E. Leg 6, anterior surface.
874
and from Misophriopsis in the presence of an outer
seta on the proximal exopod segment of the female
fifth leg.
ACKNOWLEDGMENTS
We express our sincere thanks to the captain and crew
of the T/RV Toyoshio-maru of Hiroshima University for
cooperation at sea. Thanks are due to Prof. E. Harada of
the Seto Marine Biological Laboratory for his permission
to observe the specimens of the present new misophrioid
copepod which the late Dr. T. Ito had been examining
just before his death. This work was in part supported by
grants from the Research Institute of Marine Inverte-
brates and the Nissan Science Foundation to one of the
authors (SO).
REFERENCES
1 Ohtsuka, S. and Mitsuzumi, C. (1990) A new asym-
metrical near-bottom calanoid copepod, Paramis-
ophria platysoma, with observations of its in-
tegumental organs, behavior and in-situ feeding
habit. Bull. Plankton Soc. Japan, 36: 87-101.
2 Ohtsuka, S., Fosshagen, A. and Go, A. (1991) The
hyperbenthic calanoid copepod Paramisophria from
Okinawa, South Japan. Zool. Sci., 8: 793-804.
3
S. OutTsuKA, R. Huys et al.
Huys, R., Ohtsuka, S., Boxshall, G. A. and Ito, T.
(1992) Itoitantulus misophricola gen. et sp. nov.: first
record of Tantulocarida (Crustacea: Maxillopoda) in
the North Pacific region. Zool. Sci., 9: 877-888.
Boxshall, G. A. (1983) Three new genera of mis-
ophrioid copepods from the near-bottom plankton
community in the North Atlantic Ocean. Bull. Br.
Mus. Nat. Hist. (Zool.), 44: 103-124.
Boxshall, G. A. (1989) Colonization of inland marine
caves by misophrioid copepods. J. Zool. Lond., 219:
521-526.
Huys, R. and Boxshall, G. A. (1991) Copepod
Evolution, The Ray Society, London, 468 pp.
Boxshall, G. A. and Iliffe, T. M. (1987) Three new
genera and five new species of misophrioid copepods
(Crustacea) from anchialine caves on Indo-West
Pacific and North Atlantic islands. Zool. J. Linnean
Soc., 91: 223-252.
Boxshall, G. A. (1990) A new species of Misophria
(Copepoda: Misophrioida) from Hong Kong. In
“Proceedings of the Second International Marine
Biological Workshop: The Marine Flora and Fauna
of Hong Kong and Southern China, Hong Kong,
1986”. Ed. by B. Morton, Hong Kong University
Press, Hong Kong, pp. 515-522.
Sars, G. O. (1903) An account of the Crustacea of
Norway V. Copepoda Harpacticoida. Bergen
Museum, pp. 1-28.
ZOOLOGICAL SCIENCE 9: 875-886 (1992)
© 1992 Zoological Society of Japan
Itoitantulus misophricola gen. et sp. nov.: First Record of
Tantulocarida (Crustacea: Maxillopoda)
in the North Pacific Region
Rony Huys!, Susumu OntsukA*, GEOFFREY A. BOXSHALL!
and Tatsunort IT6°
'Department of Zoology, The Natural History Museum, Cromwell Road, London
SW7 SBD, England, * Fisheries Laboratory, Hiroshima University, Takehara,
Hiroshima 725, Japan, and 3The Seto Marine Biological Laboratory,
Kyoto University, Wakayama 649-22, Japan
ABSTRACT—A new tantulocaridan, Itoitantulus misophricola Huys, Ohtsuka et Boxshall gen. et sp.
nov., (Crustacea) ectoparasitic on a hyperbenthic misophrioid copepod, Misophriopsis okinawensis
Ohtsuka, Huys, Boxshall et It6, 1992, is described from Kume Island, Okinawa, South Japan. This is
the first record of Tantulocarida from the North Pacific region. The new tantulocaridan is placed in the
Deoterthridae on account of the 1-segmented abdomen, the absence of a rostrum in the tantulus larva,
the segmentation of the rami of the thoracopods, and the position of the expanded trunk sac in the male.
The new genus can be distinguished from other deoterthrid genera by the absence of a lobate endite of
thoracopod 1 and the presence of the dorsally directed, recurved spine on the apex of the sixth
thoracopod in the tantulus larva.
INTRODUCTION
The Tantulocarida was established as a new class
of the subphylum Crustacea by Boxshall and Lin-
coln [1]. The recent phylogenetic analysis by
Boxshall and Huys [2] revealed that the Tantulo-
carida is a taxon within the class Maxillopoda and
closely related to the Thecostraca, Branchiura and
Ostracoda in having an unpaired penis derived
from the seventh thoracic legs of the adult male.
The maxillopodan affinities of the Tantulocarida
were supported by Huys’ [3] new interpretation of
the trunk tagmosis of tantulocarids. All known
species of the Tantulocarida are ectoparasitic on
crustacean hosts, including harpacticoid copepods,
myodocopid ostracods, asellote isopods, cuma-
ceans, and tanaidaceans. More than 20 species
have been described from the subtidal to the
abyssal depth zones in the North and South Atlan-
tic, the Indian and the South Pacific Oceans [1-
Accepted May 28, 1992
Received January 31, 1992
2 To whom all correspondence should be addressed.
3 Deceased on April 8th, 1990
15]. The hypothetical life cycle of the Tantulocar-
ida was presented by Boxshall and Lincoln [8] and
Huys [3]. It is characterized by extreme abbrevia-
tion: tantulocaridans have a single larval stage
which attaches to a crustacean host, the female
then sloughs its pedigerous and abdominal somites
and forms a sac-like trunk containing eggs behind
the cephalic shield, whereas the male differentiates
within the expanded larval thoracic somites and is
released as a free-swimming stage with six pairs of
powerful thoracic legs for swimming but no mouth-
parts.
A new species of the Tantulocarida infested a
new species of the Misophrioida collected from the
near-bottom off Kume Island, Okinawa, South
Japan (see [16]). This is the first record of a
tantulocaridan in the North Pacific Ocean and the
first of a tantulocaridan parasitic on misophrioid
copepods. The present paper describes immature
females and males, and the tantulus larva of the
new tantulocaridan from Okinawa.
One of the authors, Tatsunori It6 deceased
suddenly on April 8, 1990 at the age of 45 before
completion of this study. The first three authors
876 R. Huys, S. OutsuKA et al.
(RH, SO, GAB), therefore, determined on car-
rying out his intention and dedicate this study to
the late Dr. T. Ito.
MATERIALS AND METHODS
The host misophrioid copepod Misophriopsis
okinawensis Ohtsuka, Huys, Boxshall et It6, 1992
[16] was collected at a depth of about 167 m off
Kume Island, Okinawa, South Japan (26°17.9'N,
126°54.2°E) on 23 May 1989 (local time: 0824—
0858). The sampling gears and techniques and
laboratory procedures were described in detail in
Ohtsuka et al. [16]. All types of the new species of
the Tantulocarida are deposited in the collections
of The Natural History Museum, London.
Family Deoterthridae Boxshall et Lincoln
Genus /toitantulus Huys, Ohtsuka et
Boxshall gen. nov.
Diagnosis. Yantulus larva comprising cepha-
lon, 6 pedigerous somites and 2-segmented uro-
some. First thoracic tergite largely concealed
beneath posterior margin of dorsal cephalic shield.
Cephalic shield tapering anteriorly, giving a
pointed appearance; ornamentation consisting of 4
anterolateral, 2 dorsal and 6 posterior pairs of
pores and 2 distinct longitudinal lamellae. Cepha-
lic stylet straight. Thoracopods 2 to 5 with well
developed endite, thoracopods 1 and 6 without
endite. Protopods of thoracopods 1 to 5 undi-
vided; exopods apparently 2-segmented with 2 (leg
1), 4 (legs 2-4) or 5 setae (leg 5). Setae of legs 2 to
5 on distal exopod segment, leg 1 with 1 seta on
each segment. Endopod apparentlyl-segmented,
lacking setae in leg 1 or armed with 2 setae (legs 2—
5). Thoracopod 6 without distinct rami, armed
with 2 setae apically, the medial seta very strong
and curved. Posterior urosomal somite
ornamented with 5 incomplete transverse lamellae
and an array of spinous processes around posterior
margin. Caudal rami armed with 2 long, distinctly
geniculate setae and a shorter simple seta. Trunk
sac containing adult male formed posterior to 6th
thoracic tergite. Male cephalothorax with 4 pairs
of aesthetascs anteriorly. Penis slightly recurved.
Caudal rami with 3 setae. Adult female with scar
formed after larval trunk sloughed located some
distance posterior to ventral margin of head. Ecto-
parasitic on a misophrioid copepod Misophriopsis
okinawensis Ohtsuka, Huys, Boxshall et It6, 1992.
Type species. Itoitantulus misophricola Huys,
Ohtsuka et Boxshall gen. et sp. nov.
Etymology. The generic name “J/toitantulus” is
named in honor of the late Dr. Tatsunori It6, and
derived from his family name, It6 and the Latin
tantulus, which forms part of the name Tantulo-
carida.
Itoitantulus misophricola Huys, Ohtsuka
et Boxshall gen. et sp. nov.
(Figs. 1-7)
Types. All types infesting Misophriopsis oki-
nawensis Ohtsuka, Huys, Boxshall et It6, 1992
collected from the sandy bottom (ca. 167m in
depth) off Kume Island, Okinawa, South Japan,
and deposited in the Natural History Museum,
London. Holotype: tantulus larva attached on
antennary basis of M. okinawensis. BM (NH)
1991. 183. Paratypes: 3 tantulus larvae, 2 early
stage males, 4 early stage females, BM (NH) 1991.
184-192.
Description. Tantulus larva (holotype). Body
comprising cephalon covered by dorsal shield, 6
pedigerous somites and 2-segmented urosome
(Fig. 1-A, B). Total body length 156 ~m, mea-
sured from tip of cephalon to posterior margin of
caudal rami. Cephalon tapering markedly towards
oral disc; posterior margin of dorsal shield slightly
convex. Shield about 2.1 times longer than wide
(78 um X37 um); 1 pair of longitudinal epicuticu-
lar lamellae extending almost to base of oral disc;
short transverse lamellae present near rear margin.
Rostrum absent. Oral disc about 13 ~m in dia-
meter, located anteroventrally at frontal margin of
head; disc bearing 1| pair of filament-like structures
laterally, originating beneath the outer sheath.
Pore pattern comprising 4 anterolateral pairs, 1 of
which with sensillum, 2 dorsal pairs and 6 postero-
lateral pairs, 2 of which with sensillum. Ventral
surface lacking ornamentation. Cephalic stylet
visible through integument, about 36 ~m long,
more or less straight, without lateral barbs, base
hollow. Little internal structure discernible.
Tantulocaridan from the North Pacific 877
Fic. 1. Jtoitantulus misophricola gen. et sp. nov. Tantulus larva (holotype). A. Habitus, dorsal view, tantulus larva
attached to antennary basis of the host Misophriopsis okinawensis. B. Habitus, lateral view; C. Thoracic somite
1, anterior view; D. Abdomen, dorsal view.
878
R. Huys, S. OHTSUKA et al.
| | olo2in A-D |
Fic. 2.
Itoitantulus misophricola gen. et sp. nov. Tantulus larva (holotype). A. Thoracopod 1; B. Thoracopod 2; C.
Thoracopod 5; D. Thoracopod 6, seventh thoracic somite and abdomen, ventral view.
Tantulocaridan from the North Pacific 879
Fic. 3. Jtoitantulus misophricola gen. et sp. nov. Male contained within trunk sac formed by the preceding tantulus
stage (paratypes). A. Early stage male attached to second exopodal segment of antenna of Misophriopsis
okinawensis (adult female); B. Early stage male attached to antennule of M. okinawensis (copepodid V female);
C. Early stage male more developed than the stage illustrated in A, enlargement of B, lateral view; D. Same,
dorsal view; E. Lateral view of head of early stage male attached to antennulary segment of the host M.
okinawensis, showing internal anatomy.
880 R. Huys, S. OnTSUKA et al.
Fic. 4. Itoitantulus misophricola gen. et sp. nov. Early stage male (A) and female (B-D) (paratypes). A. Recurved
penis (indicated by an arrowhead) and abdomen of early stage male (see Fig. 3-C), lateral view; B. Early stage
female attached to antennulary segment of Misophriopsis okinawensis, dorsal view; C. Early stage female, lateral
view, scar indicated by an arrowhead; D. Early stage female more developed than the stage illustrated in C,
lateral view, scar indicated by an arrowhead.
Tantulocaridan from the North Pacific 881
Fic. 5.
Itoitantulus misophricola gen. et sp. nov. SEM micrographs of tantulus larva. A. Habitus, lateral view,
attached to antenna of Misophriopsis okinawensis; B. Isolated head attached to antenna of host, dorsolateral
view; C. Isolated head attached to antennule of host, dorsal view; D. Isolated head, posteroventral view, sensilla
indicated by large arrows, pores with collar by small arrows. Scale bars=10 um.
Thoracic somites 1 to 6 each with well developed
tergite, that of 1st somite (Fig. 1-C) small, largely
concealed beneath posterior rim of cephalic shield;
3rd to 5th tergites ornamented with 4 longitudinal
lamellae. Each somite with well developed tho-
racopod. Thoracopod 1 (Fig. 2-A) comprising un-
divided protopod, 2-segmented exopod and 1-
segmented endopod; protopod without endite,
armed with single medial spine; endopod with bifid
tip; exopod with short lateral seta on Ist segment
and long apical seta on 2nd segment. Thoracopods
2 to 4 (Fig. 2-B) comprising undivided protopod
bearing endite armed with 2 coupling spines, 2-
segmented exopod and 1-segmented endopod; ex-
opod with 2 long and 2 medium length setae on
distal segment; endopod with bifid tip, armed with
2 setae arising from common base located laterally
on proximal swollen part of ramus. Thoracopod 5
(Fig. 2-C) as in thoracopods 2 to 4 but with 2 long
and 3 short setae on exopod. Sixth thoracopod
(Fig. 2-D) with undivided protopod bearing 2 cou-
pling spines medially; 2 apical setae representing
rami, lateral seta slender and straight, powerful
medial seta strongly recurved dorsally.
Urosome (Figs. 1-D, 2-D) 32 um in length, com-
prising small 7th thoracic somite and large ab-
dominal somite. Seventh thoracic somite un-
ornamented. Abdominal somite 1.5 times longer
than wide (25 ~m X16 ~m); ornamented with 5
transverse epicuticular lamellae, 2 of which are
882 R. Huys, S. OntTsuKA et al.
Fic. 6.
Itoitantulus misophricola gen. et sp. nov. SEM micrographs of tantulus larva. A. Oral disc, dorsal view; B.
Oral disc, dorsolateral view, folded anterior part of cephalic shield indicated by an arrow; C. Cephalic shield of
isolated head, posterodorsal view, dorsal pores indicated by arrows; D. Dorsal pores indicated by arrows. Scale
bars=10 um (A, C); 1 xm (B, D).
incomplete ventrally; posterior margin with 3 pairs
of spinous processes ventrally, decreasing in size
away from midline. Caudal rami not clearly delim-
ited from somite; bearing 2 pairs of large setae,
each with transverse flexure plane proximally and
a complex, sheathed geniculation more distally;
3rd pair of simple setae present, arising dorsal to
main caudal setae, either side of operculum-like
structure with finely spinulated free margin.
Male metamorphosis (paratypes). Adult male
formed in reflexed position within expanded trunk
sac of preceding tantulus larva (Fig. 3-A~D).
Tantulocaridan from the North Pacific 883
: be
fis) j FG
Fic. 7. Jtoitantulus misophricola gen. et sp. nov. SEM micrographs of tantulus larva. A. Thoracic somites and
urosome, lateral view; B. Thoracic somites, enlargement of A, tergopleural grooves indicated by arrows; C.
Thoracopod 6 and urosome, enlargement of A, lateral view; D. Thoracopods 1 to 4. Scale bars=10 um (A, B,
D); 1 um (C).
Trunk sac located dorsally, posterior to 6th tho-
racic tergite. Head, rest of thorax and urosome
remaining unchanged from preceding stage; 1st
tergite beneath cephalic shiled. Metamorphosing
male supplied with nutrients via tissue connection
(umbilical cord) with larval head (Fig. 3-E). Male
at early stage in material available; thoracopods
1-6 present, setose, details not visible; cepha-
884 R. Huys, S. Ontsuka et al.
lothorax with 4 pairs of aesthetascs (Fig. 3-C).
Penis recurved at early stage of development
(arrowed in Fig. 4-A). Caudal ramus armed with 3
short spiniform elements (Fig. 4-A).
Adult female (paratypes). Early stages of adult
female showing scar formed by sloughing of larval
trunk (arrowed in Fig. 4-C, D). Larval head
unchanged from preceding stage (Fig. 4-B~D).
Trunk sac contents undifferentiated at this early
stage.
Etymology. The specific name “misophricola”
is derived from the host misophrioid copepod, and
the Lation colere, meaning to inhabit.
SEM observations of tantulus larvae of
Itoitantulus misophricola Huys, Ohtsuka
et Boxshall gen. et sp. nov.
Tantulus larvae of Jtoitantulus misophricola
were also examined with SEM (Figs. 5~7). The
attachment sites of the examined tantulus larvae
were on proximal segments of the antennules (Fig.
5-C) and the basis and rami of the antennae (Fig.
5-A, B) of the host misophrioid, Misophriopsis
okinawensis. Isolated cephalic shields without
thoracic somites and abdomen were often found
remaining on the host (Fig. 5-B~D), probably
due to damage during collection. The oral disc are
shown in Figs. 5-C and 6-A, B. The oval oral disc
is ca. 8 wm in length and ca. 5 um in width. The
cephalic shield has numerous integumental pores
and sensilla and has complex epicuticular orna-
mentation. The integumental organs are basically
distributed symmetrically except for two dorsome-
dial pores with a complex opening. The posterior
margin of head (Fig. 5-D) has two pairs of fine
sensilla (indicated by large arrows) and a pair of
relatively large pores with collar (indicated by
small arrows). The collar seems to function as an
operculum of the pore, because some of pores are
closed by the collars (see Figs. 5-D, 6-C), but this
may be an artifact of the preparation process for
SEM. Such integumental organs are hitherto
unknown in any other tantulocaridan species [1-
15]. The dorsal surface of head is entirely covered
with fine mesh-like epicuticular ridges (Fig. 6-C,
D), while the ventral surface has no epicuticular
ridges such as on the dorsal surface but is smooth
(Fig. 5-D). A pair of longitudinal lamellar plates
on the dorsomedial surface extends from near the
oral disc to in front of the pores with a complex
opening (Figs. 5-C, 6-C). These complicated
epicuticular structures on the cephalic shield are
also found in tantulus larvae of other genera,
Campyloxiphos, Coralliotantulus and Apho-
tocentor, but not in Microdajus [3, 10, 12, 14, 15].
Thoracic somites lack mesh-like epicuticular
ornamentation (Fig. 7-A, B), but each possesses a
transverse lamellar ridge dorsally, and a pair of
pleurae laterally separated by a deep tergopleural
groove (Fig. 7-B, indicated by an arrow). Such
deep tergopleural grooves on thoracic somites are
also found in Doryphallophora harrisoni (Boxshall
and Lincoln, 1987) [8].
The urosome is depicted in Fig. 7-C. The joint is
between the seventh thoracic and first abdominal
somites. The seventh thoracic somite is not
ornamented. The abdomen is partly covered with
small epicuticular prominences as in thoracopod 6.
The caudal ramus is ornamented with serrated
processes on the posterior end.
Thoracopods 1 to 4 and thoracopod 6 are shown
in Fig. 7-D and -C, respectively. The outermost
setae on apical exopod segments of thoracopods 1
to 4 are clearly found to be serrate. The curved,
outer spiniform seta on thoracopod 6 bears minute
spinules proximally and medially, and the lateral
seta is also serrate.
DISCUSSION
The new tantulocaridan definitely belongs to the
family Deoterthridae redefined by Huys [15], on
the basis of 2-segmented urosome in the tantulus
larvae, the absence of a rostrum from the tantulus,
the segmentation of the rami of thoracopods, and
the position of the expanded trunk sac in the male
behind the sixth thoracic tergite. The Deoterthri-
dae currently comprises six genera, Deoterthron
Bradford et Hewitt, 1980, Boreotantulus Huys et
Boxshall, 1988, Dicrotrichura Huys, 1989, Apho-
tocentor Huys, 1990, Campyloxiphos Huys, 1990
and Coralliotantulus Huys, 1990 [3, 15]. The new
genus can be distinguished from all of these by the
absence of a lobate endite from thoracopod 1.
Another distinctive feature is the presence of the
Tantulocaridan from the North Pacific 885
dorsally-directed, recurved spine on the apex of
the sixth thoracopod in the tantulus larva. This is
not found in any other tantulocaridans although
the form of the sixth leg of Dicrotrichura is similar.
Within the Deoterthridae the affinities of [toitantu-
lus clearly lie with Dicrotrichura, with which it
shares the modified caudal setae, an unarmed
endopod on thoracopod 1 and a recurved medial
seta on the sixth thoracopod (see [13]).
Itoitantulus misophricola is the first occurrence
of the Tantulocarida in the North Pacific Ocean,
although six species have hitherto been recorded
from the shallow- to deep-seas around Tasmania,
New Zealand and Peru [3]. As already shown by
Huys [3], tantulocaridans are widely distributed
from high latitudes to subtropical regions in both
northern and southern hemispheres. The discov-
ery of Coralliotantulus coomansi Huys, 1990 from
the Great Barrier Reef [14] might have suggested a
new locality of tantulocaridans in the subtropical
regions in Japanese waters. Tantulocaridans are
usually found parasitic on shallow- and deep-water
epibenthic crustaceans but rarely on hyperbenthic
or pelagic crustaceans except for the new tantulo-
caridans. This may suggest the epibenthic origin of
ancestral tantulocaridans.
This is the first record of tantulocaridans parasit-
ic On misophrioid copepods. The Deoterthridae
currently accommodates seven species, four of
which parasitized harpacticoid copepods, one
parasitized ostracods and the rest were found free
in sediment before attachment to hosts (see [3]).
The present new tantulocaridan infested a variety
of body parts of the host misophrioid copepods,
namely, the antennules, antennae, mandibular
palps, maxillipeds, and urosomes, but was most
usually found attached to the antennae. Four
deoterthrids parasitic on harpacticoids, namely,
Deoterthron lincolni (Boxshall, 1988), Boreotantu-
lus kunzi Huys et Boxshall, 1988, Campyloxiphos
dineti Huys, 1990, and Coralliotantulus coomansi
Huys, 1990 were attached to cephalosomes, uro-
somal somites, swimming legs and caudal setae of
their hosts [9, 11, 14, 15]. Deoterthron dentatum
Bradford et Hewitt, 1980 was found attached to a
seta on the antennary exopod of ostracod [6, 15].
Within the Deoterthridae, only the present new
tantulocaridan and D. dentatum have been found
infesting the cephalic appendages of their hosts.
ACKNOWLEDGMENTS
We express our sincere thanks to the captain and crew
of the T/RV Toyoshio-maru of Hiroshima University for
cooperation at sea. We are also thankful to Prof. E.
Harada of the Seto Marine Biological Laboratory for
permitting us to observe the specimens of the present
new tantulocaridan which the late Dr. T. It6 was describ-
ing just before his death. This work was in part sup-
ported by grants from the Research Institute of Marine
Invertebrates and the Nissan Science Foundation to one
of the authors (SO).
REFERENCES
1 Boxshall, G. A. and Lincoln, R. J. (1983) Tantulo-
carida, a new class of Crustacea ectoparasitic on
other crustaceans. J. Crust. Biol., 3: 1-16.
2 Boxshall, G. A. and Huys, R. (1989) New tantulo-
carid, Stygotantulus stocki, parasitic on harpacticoid
copepods, with an analysis of the phylogenetic rela-
tionships within the Maxillopoda. J. Crust. Biol., 9:
126-140.
3 Huys, R. (1991) Tantulocarida (Crustacea: Maxillo-
poda): A new taxon from the temporary meioben-
thos. P. S. Z. N. I.: Mar. Ecol., 12: 1-34.
4 Greve, L. (1965) A new epicaridean from western
Norway, parasitie on Tanaidacea. Sarsia, 20: 15-19.
5 Becker, K.-H. (1975) Basipodella harpacticola n.
gen., n. sp. (Crustacea, Copepoda). Helgolander
Wiss. Meeresunters., 27: 96-100.
6 Bradford, J. M. and Hewitt, G. C. (1980) A new
maxillopodan crustacean, parasitic on a myodocopid
ostracod. Crustaceana, 38: 67-72.
7 Lincoln, R. J. and Boxshall, G. A. (1983) A new
species of Deoterthron (Crustacea: Tantulocarida)
ectoparasitic on a deep-sea asellote from New Zea-
land. J. Nat. Hist., 17: 881-889.
8 Boxshall, G. A. and Lincoln, R. J. (1987) The life
cycle of the Tantulocarida (Crustacea). Phil. Trans.
R. Soc. Lond. B315: 267-303.
9 Boxshall, G. A. (1988) A new genus of tantulocar-
idan (Crustacea: Tantulocarida) parasitic on a har-
pacticoid copepod from Tasmania. Bull. Br. Mus.
Nat. Hist. (Zool.), 54: 271-274.
10 Grygier, M. J. and Sieg, J. (1988) Microdajus
(Crustacea: Tantulocarida) parasitic on an Antarctic
tanaidancean, and a range extension of M. langi
Greve. J. Nat. Hist., 22: 1495-1505.
11 MHluys, R. and Boxshall, G. A. (1988) A new genus
and species of tantulocaridan (Crustancea: Tantulo-
carida) parasitic on a harpacticoid copepod from the
Skagerrak. Sarsia 73: 205-211.
12
13
14
886
Boxshall, G. A., Huys, R. and Lincoln, R. J. (1989)
A new species of Microdajus (Crustacea: Tantulo-
carida) parasitic on a tanaid in the northeastern
Atlantic, with observations on M. langi Greve. Syst.
Parasit., 14: 17-30.
Huys, R. (1989) Dicrotrichura tricincta gen. et spec.
nov.: A new tantulocaridan (Crustacea: Maxillopo-
da) from the Mediterranean deep sea off Corsica.
Bijdr. Dierk., 59: 243-249.
Huys, R. (1990) Coralliotantulus coomansi gen. et
sp. n.: first record of a tantulocaridan (Crustacea:
Maxillipoda) from shallow subtidal sands in tropical
15
16
R. Huys, S. OuntsuKa et al.
waters. Stygologia, 5: 183-198.
Huys, R. (1990) Campyloxiphos dineti gen. et spec.
nov. from off Namibia and a redefinition of the
Deoterthridae Boxshall & Lincoln (Crustacea: Tan-
tulocarida). J. Nat. Hist., 24: 415-432.
Ohtsuka, S. Huys, R., Boxshall, G. A. and Ito, T.
(1992) Misophriopsis okinawensis sp. nov. (Crus-
tacea: Copepoda) from hyperbenthic waters off Oki-
nawa, South Japan, with definitions of related
genera Misophria Boeck, 1864 and Stygomisophria
gen. nov. Zool. Sci., 9: 861-876.
ZOOLOGICAL SCIENCE 9: 887-889 (1992)
[COMMUNICATION]
© 1992 Zoological Society of Japan
Effect of Monochromatic Illumination of the Brain on the
Phototactic Behavior of Orb Weaving Spiders,
Argiope amoena and Nephila clavata
SHIGEKI YAMASHITA
Biological Laboratory, Kyushu Institute of Design,
Shiobaru, Fukuoka 815, Japan
ABSTRACT—The effect of monochromatic light ap-
plied to the brain on the phototactic behavior of tethered
orb weaving spiders was examined. It is suggested that
cerebral photosensitive neurons which have a maximum
sensitivity at about 440 nm play a role in phototactic
behavior.
INTRODUCTION
The existence of photosensitive neurons in the
brain of orb weaving spiders has been revealed
electrophysiologically [1-3] and behaviorally [4,
5]. Yamashita and Tuji [4] examined the phototac-
tic behavior of tethered orb weaving spiders,
Argiope and Nephila, while they walked on a
y-maze globe. On a dark background, the spiders
tended to turn away from light given to the eyes.
On a light background, however, the spiders
tended to turn towards the light. When the brain
was illuminated through the cuticle covering it with
a small light spot, the spiders also tended to turn
towards the light given to the eyes. Blue light
given to the brain had a marked effect on the
phototactic respone, but not green light. Yamashi-
ta and Tuji [4] concluded that cerebral phtosensi-
tive neurons control phototactic behavior, and
suggested that these neurons had maximum sensi-
tivity in the blue region. In the present study, I
examined the efficiency of monochromatic light
given to the brain on phototactic behavior.
Accepted June 5, 1992
Received April 30, 1992
MATERIALS AND METHODS
Female orb weaving spiders, Argiope amoena
and Nephila clavata, were collected in open fields.
The methods for these experiments were similar to
those described previously [4]. In brief, the spider
was held rigidly in space and was given a y-maze
globe to hold (Fig. 1). For illumination of the
eyes, two light beams emitted from two tungsten
filament lamps were focused onto the ends of two
light guides. The exits of the light guides were
LG
to left eyes
toright eyes
=
Fic. 1. The spider is fixed in space by a quartz light
guide and given a y-maze globe to hold. LG, light
guide.
888 S. YAMASHITA
aimed horizontally at the right eyes from 45°
clockwise and at the left eyes from 45° anticlock-
wise to the body axis of the spider, respectively.
The intensity of the light was about 30 lux at the
eyes. For monochromatic light stimulation of the
brain, a light beam emitted by a 500 W Xenon arc
lamp was passed through interference filters and
focused onto one end of a quartz light guide of 2
mm in diameter. The other end of the light guide
was joined onto the central part of the prosoma
just above the brain (Fig. 1). The intensity of the
light given to the brain (I,,) is referred to as
relative quantum flux. Maximum relative intensity
(Ip,=1.0) corresponded to about 10'* quanta/
cm*-sec at the output of the light guide.
In most cases, spiders made 100 turns at y-arms
for each test (e.g. each point on Figure 2); 50
during stimulation of the left eyes and 50 during
stimulation of the right eyes. Prior to the start of
each test run, spiders were adapted to each test
condition for 5-10 min. Usually, three or four
tests were performed on a single spider. The
turning reaction (TR) was defined as P—N/P+N,
where P is the number of turns at y-arms directed
towards the light given to the eyes, and N is the
number of turns directed away from the light.
+0.2
Turning reaction
-0.4
_——————E—— ee
0 0.2 0.4 0.6 0.8 1.0
Illumination of brain, Ipr
Fic. 2. Effects of illumination of the brain by 360, 440
and 520nm light on phototactic response. The
turning reactions (TR=P—N/P+N) are plotted
against the relative intensity of light given to the
brain (1,,).
RESULTS AND DISCUSSION
The effects of intensity of 360, 440 and 520 nm
light given to the brain on the phototactic response
are shown in Figure 2. The three curves were
obtained from three different spiders. When the
intensity of light given to the brain (I,,) increases
from 0 to 1.0, the turning reactions (TRs) increase
by 0.54 from —0.36 to +0.18 for 440 nm, by 0.28
from —0.42 to —0.14 for 360 nm and by 0.08 from
—0.28 to —0.20 for 520nm light. Cerebral
illumination by 440 nm light changes TR from
negative to positive but dose not do so in the cases
of 360 and 520 nm light, at least, in the region of
the intensity used in the present study. It is clear
that blue light has a marked effect on phtotactic
response, as was reported by Yamashita and Tuji
[4].
The efficiency of monochromatic light given to
the brain was defined, for convenience, as the
difference between TR at I,,=1.0 and TR at I,,=
0. Spectral efficiencies for 1-3 wavelengths were
determined for each spider. The average spectral
efficiency curve obtained from 18 spiders is shown
in Figure 3. The curve has a single peak at 440 nm.
The mean value of TRs at I,,=0 for these spiders
was —0.38+0.10 (SD). The cerebral photosensi-
tive neurons reported by Yamashita and Tateda [1]
had a maximum sensitivity at 420-440 nm, sug-
gesting that these neurons control the phototactic
behavior of the spiders.
0.6
o
bh
Efficiency
i)
N
440 480 520 560
Wavelength, nm
360 400
Fic. 3. Average spectral efficiency curve. See text.
Vertical lines indicate standard deviation.
ACKNOWLEDGMENTS
This research was supported in part by Grant 03640608
from the Ministry of Education of Japan.
Phototactic Behavior of Spiders 889
REFERENCES
1 Yamashita, S. and Tateda, H. (1983) J. Comp.
Physiol., 150: 467-472.
2 Yamashita, S. (1985) In “Neurobiology of Arach-
3
4
5
nids”. Ed. by F. G. Barth, Springer-Verlag, Berlin-
Heidelberg-New York-Tokyo, pp. 103-117.
Yamashita, S. (1990) Vision Res., 30: 817-821.
Yamashita, S. and Tuji, R. (1987) Zool. Sci., 4: 23-
30.
Yamashita, S. (1987) Zool. Sci., 4: 31-35.
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ZOOLOGICAL SCIENCE 9: 891-895 (1992)
[COMMUNICATION]
© 1992 Zoological Society of Japan
Isolation of Intact Yolk Spheres of Fish Embryos, Which
Contain the Majority of Lysosomal Acid Phosphatase
Responsible for Yolk Phosphoprotein Metabolism
MasAHirRO MuraKamt., Icuiro Iucui and
KENJIRO YAMAGAMI-
Life Science Institute, Sophia University, 7-1 Kioicho,
Chiyoda-ku, Tokyo 102, Japan
ABSTRACT — Intact yolk spheres (membrane-bounded
yolk masses) completely devoid of yolk cytoplasmic layer
(YCL), yolk syncytial layer (YSL) and a blastoderm
could be isolated from mid-blastulae or early gastrulae of
the teleost, Oryzias latipes. Most of soluble yolk proteins
and acid phosphatase in a whole embryo were found in
the isolated yolk sphere. More than 80% of the acid
phosphatase in the yolk sphere were sensitive to tarta-
rate, probably belonging to lysosomal acid phosphatase.
Considering that the acid phosphatase in the early
embryos of this fish is responsible for dephosphorylation
of yolk phosphoproteins [1-4], the present result strong-
ly suggests that yolk phosphoproteins are dephosphory-
lated in the yolk sphere itself independently of the
surrounding YCL, YSL and blastoderm.
INTRODUCTION
In oviparous vertebrates, yolk phosphoproteins
provide the embryo with amino acids (or peptides)
and phosphates as nutrients. Recently. we iden-
tified two phosvitins and two lipovitellins in egg
yolk of the fish, Oryzias latipes [4, 5]. A remark-
able degradation of these phosphoproteins occurs
during early phase of development from blastulae
(Day 1 embryos) to optic vesicle stage embryos
(Day 2 embryos) and the degradation is considered
Accepted May 19, 1992
Received April 14, 1992
' Present address: Division of Radiation Hazards,
National Institute of Radiological Sciences, Chiba
263, Japan
* To whom correspondence should be addressed.
to be closely associated with dephosphorylation by
acid phosphatase and proteolysis by some sub-
cellular structure-associated cathepsin-like pro-
tease [1-4]. On the other hand, an important role
of periblast, or yolk syncytial layer (YSL) [6], in
yolk nutrition is conjectured [7]. In Oryzias latipes
embryos, total amount of soluble yolk proteins
decreases markedly after Day 3 instead of before
Day 2 of development [4], and electron micros-
copy reveals that various-sized yolk masses are
endocytosed actively by YSL only after Day 3
(Murakami et al., unpublished). Thus, the dephos-
phorylation of phosphoproteins probably occurs
independently of YSL. The role of yolk sphere (a
membrane-bounded whole yolk mass) in yolk
digestion in fish has scarcely been analyzed, as
intact yolk spheres have never been isolated to be
analyzed. In the course of our Studies on egg yolk
proteins, we worked out a practical method of
isolation of intact yolk spheres completely devoid
of YSL and yolk cytoplasmic layer (YCL) from
blastulae of Oryzias latipes, and found that the
yolk spheres contained the majority of lysosomal
acid phosphatase present in whole embryos. This
method would facilitate a further analysis of yolk
metabolism in fish embryos.
MATERIALS AND METHODS
Embryos Outbred fish of orange red variety of
Oryzias latipes were fed on Tetrafin in order to
892 M. Murakami, I. IucHI AND K. YAMAGAMI
obtain naturally fertilized eggs as described else-
where [4]. Mid-blastulae and early gastrulae (Day
1 embryos) were exclusively employed as mate-
rials.
Preparation of partially purified hatching en-
zyme A mixture of partially purified high and
low choriolytic enzymes (HCE and LCE) was
obtained from the hatching liquid following the
Step 1 of the purification procedure of HCE [8].
The 0.02 M Tris-HCI-0.3 M NaCl (pH 8.5) eluate
(CM-0.3 enzyme) which showed a high activity of
egg envelope (chorion)-digestion was frozen and
stored until use. The CM-0.3 enzyme (OD2g9=
0.16) was mixed with an equal volume of 0.02 M
Tris: HCl-0.25 M sucrose (pH 7.2) and the mixture
was employed as a hatching enzyme solution.
Isolation of yolk sphere. About 50 mid-bla-
stulae or early gastrulae were incubated in 1 ml of
the hatching enzyme solution on 2% agar plate at
30°C for about 1hr. Partially digested egg en-
velopes (chorions) could be easily removed by a
pair of sharp tweezers. The dechorionated
embryos were then transferred to 0.02M Tris-
HCI-0.25 M sucrose-0.1 M ethylenediamine tetra-
acetate (EDTA) (pH7.2), and incubated at
30°C for 1 hr. Somewhat loosened YCL could be
peeled off, together with YSL and a blastoderm,
by sharp tweezers. A yolk sphere surrounded by a
fragile membrane could be washed by transferring
carefully to a buffered 0.25 M sucrose containing
no EDTA.
Electron microscopy Specimens were fixed in
ice-cold 4% paraformaldehyde-3% glutaraldehyde-
0.19% picric acid-0.2M_ sucrose-0.1M _ phos-
phate buffer (pH 7.4) for 3 hr. The fixed speci-
mens were washed with 0.2 M phosphate buffer-
0.4M sucrose (pH 7.4) for 15 min three times.
Post-fixation was performed with 1.33% OsO,-0.2
M sucrose-0.1 M phosphate buffer (pH 7.4) at 5°C
for 2.5 hr. After washing with 0.4 M sucrose-0.2 M
phosphate buffer (pH 7.4) for 15 min two times,
the fixed specimens were dehydrated through an
alcohol series, followed by treatment with pro-
pylene oxide and embedded with Spurr embedding
resin (Polyscience Inc., Warrington). The sections
were cut at 0.1 ~m, stained with lead citrate and
uranyl acetate and observed with a Hitachi H300
transmission electron microscope.
Polyacrylamide gel electrophoresis Whole
embryos or isolated yolk spheres were homoge-
nized in ice-cold 0.02 M Tris-HCI-0.25 M sucrose
(pH 7.2) and the homogenates were centrifuged at
14000 g at 4°C for 10 min to prepare the extracts.
Polyacrylamide gel disc electrophoresis (disc
PAGE) using 7% gel (pH 8.0) was performed
following the method of Williams and Reisfeld [9].
Proteins were stained with Coomassie brilliant
blue and acid phosphatase was detected by the
method of Gabriel [10]; After electrophoresis, the
gels were treated with 0.1 M acetate buffer (pH
5.0) at 4°C for 15min and incubated in the
reaction/staining medium of 0.04M _ Veronal-
acetate buffer containing 0.01% sodium a-
naphthyl phosphate as substrate, 0.16% rosanilin
and 0.16% NaNO; (pH 5.0) at 4°C overnight.
Acid phosphatase activity Acid phosphatase
activity was determined following the method of
Bessey et al. [11] by using p-nitrophenyl! phosphate
(Acid Phospha-B Test, wako Pure Chemicals,
Tokyo) as substrate. Whole embryos or isolated
yolk spheres were extracted with ice-cold 0.02 M
Tris-HCl-0.25 M sucrose (pH7.2). One tenth
milliliter of the extracts, equivalent to 1-10
embryos, was incubated with 0.5 ml of 0.25%
p-nitrophenyl phosphate-0.05 M citrate buffer (pH
4.9) at 30°C min. After the reaction was stopped
by adding 5 ml of 0.05 M NaOH, the absorbance
of the mixture at 405nm was measured. Acid
phosphatase activities in the presence and absence
of 0.01 M Na-tartarate were compared to estimate
the activity of lysosomal acid phosphatase, which
was sensitive to tartarate [12, 13]. Under the
conditions, the activity determined was found to
be proportional to the amount of the enzyme.
RESULTS AND DISCUSSION
As illustrated in Fig. la, a greater part of the
membrane-bounded yolk sphere of a blastula is
surrounded by a thin YCL. The YCL is con-
tinuous to YSL, or marginal and subblastodermal
periblasts, in animal pole region. Oil drops
Yolk Sphere of Fish Embryo 893
soot Fo AT Cm ae S oes
Fic. 1.
Isolation of intact yolk spheres from Oryzias latipes embryos.
a, A diagrammatic illustration of a
mid-blastula. B; blastoderm, O; oil drops, YS; yolk sphere, YCL; yolk cytoplasmic layer, YSL; yolk syncytial
layer. A small square represents the locale of a section shown in Fig. 1c. b, Some specimens obtained in the
process of isolation of yolk spheres. 1; dechorionated Day 1 embryos (2 mid-blastulae and 1 early gastrula), 2; a
midblastula whose YCL is in the midst of removal, 3; a mid-blastula whose YCL is, together with YSL and a
blastoderm, almost removed. Oil drops are adhering to YCL and YSL in this psecimen. 4; an isolated yolk
sphere. Scale bar, 1 mm. c, An electron micrograph of a section in the locale shown in Fig. 1a as a small square.
YS; york sphere, YSL; yolk syncytial layer. d, An electron micrograph showing a surface of an isolated yolk
sphere. Arrows in c and d indicate a membrane enveloping the yolk sphere. Scale bars in c and d, 1 um.
locating in YCL are stuck in the yolk sphere at the
vegetal pole. Fig. 1b shows some specimens in the
process of isolation of intact yolk spheres from
mid-blastulae or early gastrulae. The dechorion-
ated embryos are usually posed animal pole side
down in water, as large oil drops had migrated and
attached to the vegetal pole side. On incubation in
the medium containing EDTA, the oil drops come
outward slightly and a small space appears beneath
the loosened membrane of YCL around the oil
drops. YCL can be peeled off from this space
toward animal pole with sharp tweezers under a
binocular micropscope. The oil drops are released
and float to the surface of the medium. Finally, all
YCL can be removed together with YSL and
blastoderm, and an intact yolk sphere is obtained.
894 M. Murakami, I. IucHI AND K. YAMAGAMI
a | |) a: EF 3 —s
5 j We.
1 2s 4
Fic. 2. Disc electrophoretic patterns of soluble proteins
and acid phosphatase of whole embryos (blastulae)
and yolk spheres isolated from blastulae. The
extract equivalent to one embryo or one yolk sphere
was applied to each column. 1 and 2; whole
blastula, 3 and 4; yolk sphere. 1 and 4; soluble
proteins, 2 and 3; acid phosphatase. Phosvitins are
not stained by coomassie brilliant blue [4] but are
faintly visible in the columns for phosphatase stain-
ing (x).
As shown in Fig. 1c and 1d, the yolk spheres, both
in situ and in isolation, are enveloped by a
membrane, which we can identify more clearly in
an isolated specimen. No YCL is found to be
adhering to the isolated yolk spheres. This mem-
brane has been documented for the embryos of
this fish [14] and Fundulus heteroclitus [6, 15}.
Yolk platelets in amphibian embrycs are also
surrounded by a similar membrane [16].
Disc PAGE patterns of proteins and acid phos-
phatase activity for whole embryo and the isolated
yolk sphere are shown in Fig. 2. The pattern and
density in a whole embryo are quite similar to
those in an isolated yolk sphere, indicating that the
acid phosphatase is represented by a single protein
band and that most of the acid phosphatase as well
as soluble proteins in a whole embryo are localized
to the yolk sphere. In Oryzias latipes embryos,
acid phosphatase is considered to be closely
related to dephosphorylation of yolk phosphopro-
teins during early embryonic development, as
there is a high similarity between the autolytic
dephosphorylation of yolk phosphoproteins and
the acid phosphatase activity with respect to the
pH-activity relationship and the effects of some
=)
N
0.1
=
ro)
WE
Fic. 3. Acid phosphatase activity in whole embryos and
isolated yolk spheres. The activities per two whole
midblastulae (WE) and two isolated yolk spheres
(YS) in the presence (white column) or absence
(hatched column) of 0.01M Na-tartarate are ex-
pressed as the average of 16 experiments.
YS
Acid phosphatase activity (A.os)
ions on the activity and there is an active acid
phosphatase but little alkaline phosphatase in the
early embryos [2, 3]. Thus, it is highly probable
that the acid phosphatase visualized on the disc
PAGE is responsible for dephosphorylation of
yolk phosphoproteins [2-4]. As shown in Fig. 3,
the present results indicate that about 75% of the
acid phosphatase in a whole Day 1 embryo is
localized to the yolk sphere itself. About 76% of
the activity in whole embryo and about 84% of
that of the isolated yolk sphere are sensitive to
tartarate. Therefore, a large part of the tartarate-
sensitive (or lysosomal) acid phosphatase of whole
embryo is located in the yolk sphere. The
tartarate-sensitive phosphatase are grouped into
ethanol-sensitive and -insensitive enzyme [12].
Most activity of the acid phosphatase in the yolk of
Oryzias latipes is found to be insensitive to ethanol
(Murakami ef al., unpublished). Although a
detailed mechanism of deposition of fluid yolk in
Oryzias latipes oocytes is obscure, it is highly
probable that lysosomes participate in transforma-
Yolk Sphere of Fish Embryo 895
tion of the endocytosed vitellogenin into deposited
yolk proteins (cf. [17]). The present results
directly confirm that a yolk sphere is closely
related to lysosomes in terms of the localization
and the nature of the acid phosphatase. Moreover,
it seems highly probable that dephosphorylation of
yolk phosphoproteins occurs inside the yolk
sphere.
ACKNOWLEDGMENTS
The authors wish to thank Dr. T. S. Hamazaki for his
invaluable advice on electron microscopy.
REFERENCES
1 Yamagami, K. (1960) Sci. Pap. Coll. Gen. Educ.,
Univ. Tokyo, 10: 99-108.
2 Yamagami, K. (1961) Sci. Pap. Coll. Gen. Educ.,
Univ. Tokyo, 11: 153-161.
3 Yamagami, K. (1963) Sci. Pap. Coll. Gen. Educ.,
Univ. Tokyo, 13: 223-229.
4 Murakami, M., Iuchi, I. and Yamagami, K. (1990)
Dev. Growth Diff., 32: 619-627.
5 Murakami, M., Iuchi, I. and Yamagami, K. (1991)
Comp. Biochem. Physiol., 100: 587-593.
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Zool., 206: 381-426.
Heming, T. A. and Buddington, R. K. (1988) In
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407-446.
Yasumasu, S., Iuchi, I. and Yamagami, K. (1988) J.
Biochem., 105: 204-211.
Williams, D. E. and Reisfeld, R. A. (1964) Ann. N.
Y. Acad. Sci., 121: 373-381.
Gabriel, O. (1971) In “Methods in Enzymology”.
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Bessey, O. A., Lowry, O. H. and Brock, M. J.
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Abul-Fadl, M. A. M. and King, E. J. (1949)
Biochem. J., 45: 51-60.
Dipietro, D. L. and Zengerle, F. S. (1967) J. Biol.
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10: 483-490.
Lentz, T. L. and Trinkaus, J. P. (1967) J. Cell Biol.,
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Richter, H. P. (1987) Roux’s Arch. Dev. Biol., 196:
367-371.
Wallace, R. A. (1985) In “Developmental Biology”.
Ed. by L. W. Browder. Plenum Press, N. Y., Vol. 1,
pp. 127-177.
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ZOOLOGICAL SCIENCE 9: 897-900 (1992)
[COMMUNICATION]
© 1992 Zoological Society of Japan
First Electron Microscopical Study on the Sperm Morphology
of the Sea Lily (Crinoidea, Echinodermata)
SHONAN Amemiya! and Yoko NAKAJIMA~
‘Misaki Marine Biological Station, University of Tokyo, Miura-shi,
Kanagawa 238-02, and *Department of Biology, Keio
University, Hiyoshi, Yokohama 223, Japan
ABSTRACT—The sperm morphology of the sea lily
(stalked crinoid) is described for an isocrinid species,
Metacrinus rotundus. Scanning electron microscopy
shows that the head of the sperm is spherical with an
acorn-like shape. The acrosome appears as a nipple-
shaped hemispherical protuberance on the apical tip of
the sperm head. The middle piece occupies the basal
one-third of the head, forming the “cap” of the acorn.
The sperm head is 2.0 ~m long and 1.7 «m wide, and the
acrosome area is 0.23 ~m long. The sperm flagellum is
0.15 4m thick and 43 ~m long. The sperm morphology of
the sea lily is similar to that of the feather stars.
INTRODUCTION
Sea lilies (stalked crinoids) flourished in the sea
during the Palaeozoic era, and have survived
through to the present day, keeping their morphol-
ogy essentially unchanged [1, 2]. These animals
(class Crinoidea) are the only living Pelmatozoa
among the six classes of Echinodermata, and are
considered to occupy an extremely important
position for understanding the phylogeny of the
echinoderms [1, 3]. They once had a wide range of
bathymetric distribution from shallow to deep
water, but most extant species live in seas deeper
than several hundred meters. Because of the
difficulty in obtaining live specimens, sea lilies
have been regarded for a long time as biologically
mysterious organisms [4], and details of their
gamete morphology, reproduction and develop-
ment have remained largely unknown. Recently,
Accepted June 5, 1992
Received May 6, 1992
we succeeded in obtaining sperms of an isocrinid
sea lily, Metacrinus rotundus, and observed their
morphology by scanning electron microscopy
(SEM). This report is the first to give details of the
outer features of sea lily gametes.
MATERIALS AND METHODS
Specimens of the isocrinid sea lily, Metacrinus
rotundus Carpenter, were collected by dredging
from 150m depth at Suruga bay, on the Pacific
coast of central Japan. The sperms were obtained
by dissecting the testis. Fixation of the sperms was
performed essentially by the procedure reported
by Holland and Kubota [5] with some modifica-
tions. In brief, the sperms placed on filter paper
were fixed in 2% glutaraldehyde in 70% artificial
seawater (ASW, Jamarine U, Osaka), post-fixed
in 2% osmium tetroxide in 2.5% NaHCO3, and
dehydrated with an ethanol series. The specimens
were then critical-point dried, sputter-coated with
gold and examined with a Hitachi S-510 scanning
electron microscope at 25 kV.
RESULTS AND DISCUSSION
The testes of Metacrinus rotundus were found in
the genital pinnules and in the arms at the points of
branching of the pinnules in males (Fig. 1). In
females, the ovaries were located in the same place
as the testes in males. Some fractions (if not all) of
the live sperm obtained by dissection of the testes
swam slowly, indicating that the sperm were
S. AMEMIYA AND Y. NAKAJIMA
898
Fic. 1. A part of an arm bearing some pinnules of a male isocrinid sea lily, Metacrinus rotundus, observed by
dissection microscopy. The testes (arrow-heads) are located in the genital pinnules (small arrows) and in the arm
(large arrow) at the points of branching of the genital pinnules. Bar represents 5 mm.
Fic. 2. Sperms of the isocrinid sea lily, Metacrinus rotundus, observed by scanning electron microscopy. The
acrosome (A) is surrounded by a circular groove (arrow). M, middle piece; F, flagellum. Bar represents 2 ~m.
Sea Lily Sperm 899
mature or almost mature. The head of each sperm
was almost spherical with an acorn-like shape (Fig.
2). The acrosome formed a nipple-like hemispher-
ical protuberance on the apical tip of the sperm
head. The middle piece occupied the basal
one-third of the head, forming the “cap” of the
acorn. The head size estimated from the SEM
photograph was about 2.0 vm in length with the
acrosome region measuring 0.23 um, or one-ninth
of the total head length. The width of the head
represented by the size of a nucleus which was
located between a mitochondrion and an acro-
some, was about 1.7 zm. The head size (2.0 ~m
long) was comparable to that of two other species
of sea lily (Calamocrinus diomedae and Holopus
rangii) reported by Holland [6] based on light
microscopical observations. The flagellum of the
sperm was 0.15 ~m thick and 43 um long. How-
ever, the preparation procedure for SEM observa-
tion might have caused underestimation of the
size. An oblique view around the apical tip of the
sperm head showed details of the acrosome and its
neighborhood (Fig. 2). The hemispherical nipple
of the acrosome was surrounded by a circular
groove 0.15 “m wide.
The sperm of the feather star, Florometra
serratissima [7], showed remarkable similarity in
the shape of sperm head to M. rotundus. The head
length described for F. serratissima was 2.3 um,
almost the same as that of M. rotundus. The
shapes of the middle piece and acrosome were also
very similar. The acrosome was somewhat larger
in M. rotundus than in F. serratissima and in other
echinoderm species with a spherical sperm shape
[7].
In three subphyla (Crinozoa, Asterozoa and
Echinozoa) of Echinodermata [1], two of three
Asterozoa (ophiuroids and asteroids) species
known so far have sperms with spherical heads.
The sperm morphology of the newly discovered
Asterozoa, concentricycloids, is V-shaped as in the
phoronids, and has no similarity with any other
echinoderm species [8]. Most species of holothu-
rians, one of the Echinozoa, have sperms with
spherical heads, but some species have the sperm
of cylindrical heads [7]. All sperms of another
member of the Echinozoa, the echinoids, have
conical heads [7], although the sperm heads of
some species are highly elongated [9, 10]. As to
the Crinozoa, the sperm morphology has been
examined electron microscopically for some spe-
cies of feather stars (Antedon petasus, Comanthus
japonicus and F. serratissima), and has been
reported to be spherical [7]. In contrast, Holland
[6] has reported that the sperm head of a feather
star, Antedon bifida, is conical. Information on the
sperms of sea lilies is extremely limited, and no
electron microscopical study has yet been re-
ported. Based on light microscopical studies, only
four species of sea lily have been reported so far
with regard to sperm morphology. These are
Rhizocrinus lofotensis by Ludwig [11], Bathycrinus
carpenteri by Danielsen [12], and Calamocrinus
diomedae and Holopus rangii by Holland [6].
These authors reported that the sperm heads of the
four species are spherical. The present electron
microscopical study on the sperm morphology of
M. rotundus confirmed that the sperms of this sea
lily have spherical heads. Chia et al. [7] suggested
that the spherical sperm head is the original form,
and that the sperms of many species of Echinozoa
have been modified to have conical or cylindrical
heads, whereas the sperms of Crinozoa and Aster-
ozoa have retained the original form. However,
more recent information on the sperm morphology
of concentricycloids [8] and some feather stars [6]
suggests that further analysis is necessary to
confirm the relationship between sperm morpholo-
gy and echinoderm phylogeny.
Further examination of the details of the sperm
morphology of M. rotundus, based mainly on
transmission electron microscopy, is now in prog-
ress and the findings will be reported in the near
future.
ACKNOWLEDGMENTS
We would like to thank Dr. T. Oji for his assistance to
collect the materials.
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Co. Ltd., London.
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8
S. AMEMIYA AND Y. NAKAJIMA
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D evel O m en { Published Bimonthly by the Japanese Society of
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Distributed by Business Center for Academic
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Papers in Vol. 34, No. 4. (August 1992)
40. H. Fang, T. Sakuma and K. Yanagisawa: Ammonia Determines the Alternative Pathways of
Sexual or Asexual Development in the Cellular Slime Mold Dictyostelium discoideum
41. K. Mitsunaga-Nakatsubo, A. Fujiwara and I. Yasumasu: Change in the Activity of Na‘,
K*-ATPase in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus, during Early
Development
42. H. Nakamura and N. Itasaki: Expression of en in the Prosencephalon Heterotopically
Transplanted into the Mesencephalon
43. H. Dupont, P. Deparis, M. Tujague, D. Saboulard, C. Ardourel, C. Daguzan and J.-C.
Beetschen: Differential Protein Distribution Related to Dorsoventral Polarity in Pleurodeles
waltl Cleaving Egg
44. K. Hoshino, T. Shimizu, Y. Sendai, T. Harumi and N. Suzuki: Differential Effects of the Egg
Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca** and pH in Sea Urchin
Spermatozoa
45. C. Inoue, M. Kiyomoto and H. Shirai: Germ Cell Differentiation in Starfish: The Posterior
Enterocoel as the Origin of Germ Cells in Asterina pectinifera
46. A. Shinagawa: Relative Timing of Stiffening with Various Combinations of Nucleate and
Enucleated Egg Fragments of Xenopus laevis
47. K. Kawamura and N. Yamashiki: The Determination of Spindle Polarity in Early Mitotic
Stages of the Dividing Grasshopper Neuroblasts
48. K. Hara, S. Fujiwara and K. Kawamura: Retinoic Acid can Induce a Secondary Axis in
Developing Buds of a Colonial Ascidian, Polyandrocarpa misakiensis
49. R. Yanagimachi, G. N. Cherr, M. C. Pillai and J. D. Baldwin: Factors Controlling Sperm
Entry into the Micropyles of Salmonid and Herring Eggs
50. S. Fujiwara and K. Kawamura: Ascidian Budding as a Transdifferentiation-Like System:
Multipotent Epithelium is not Undifferentiated
51. S. Nagata, M. G. Rosenfeld and K. Inoue: Development of Prolactin and Growth Hormone
Production in the Fetal Rat Pituitary: An Immunochemical Study
52. L.-N. Wei, C.-H. Lee, S.-L. Chang and Y.-S. Chu: Pathogenesis in Transgenic Mice
Expressing Bovine Cellular Retinoic Acid-Binding Protein
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(Contents continued from back cover)
of hypothalamo-pituitary-ovarian axis in the
TER, coi. RoE gOS RE eee nice en eae 791
Sawada, K. and T. Noumura: Differential
effects of testosterone and 5a-dihydrotes-
tosterone on growth in mouse submandibu-
lar gland
Morphology
Shirai, S. and K. Nakaya: Functional mor-
phology of feeding apparatus of the cookie-
cutter shark, Jsistius brasiliensis (Elasmo-
branchii, Dalatiinae)
Ando, K. and S. Arai: Neuropeptide Y in-
nervation of cerebral arteries in microchiro-
PUCKAMMD ALS ye ne alae ech us nie ane a cee avem 823
Ecology
Matsumoto, T.: Familial association, nym-
phal development and population density in
the Australian giant burrowing cockroach,
(Blattaria:
Macropanesthia __ rhinoceros
Blaberidae)
Taxonomy
Nagatomi, A.: Notes on the phylogeny of
various taxa of the orthorrhaphous
Brachycera (Insecta: Diptera) ............ 843
Ohtsuka, S., R. Huys, G. A. Boxshall and T.
It6: Misophriopsis okinawensis sp. nov.
(Crustacea: Copepoda) from hyperbenthic
waters off Okinawa, South Japan, with
definitions of related genera Misophria
boeck, 1864 and Stygomisophria gen. nov.
Huys, R., S. Ohtsuka, G. A. Boxshall and T.
It6: Itoitantulus misophricola gen. et sp.
nov.: First record of Tantulocarida (Crus-
tacea: Maxillopoda) in the North Pacific re-
AKON. ac @oeteae cet rte ca Ween a 875
ZOOLOGICAL SCIENCE
VOLUME 9 NUMBER 4
AUGUST 1992
CONTENTS
REVIEWS
Matsumoto, A.: Hormonally induced synap-
tic plasticity in the adult neuroendocrine
Bray “waccuitigasnaice sao eae ae eer 679
Bock, W. J.: The species concept in theory
and Practice .i cc. cdeac eee oo uae 697
ORIGINAL PAPERS
Physiology
Naitoh, T. and R. J. Wassersug: The emetic
response of urodele amphibians .......... 713
Miro, J. L., S. Araneda and B. Canguilhem:
Origin of serotonergic innervation of olfac-
tory bulbs in the Europian hamster, Cricetus
cricetus: An autoradiographic study using
[PH]S-HT retrograde labelling
Yamashita, S.:
mination of the brain on the phototactic
Effect of monochromatic illu-
behavior of orb weaving spiders, Argiope
amoena and Nephila clavata (COMMUNI-
CATION)
Developmental Biology
Endo, K., S. Ueno, M. Matsufuji and Y.
Kakuo: Photoperiodic control of the deter-
mination of two different seasonal diphen-
isms of the Asian comma butterfly, Polygo-
nia c-aureum L.
Ukeshima, A.: Scanning electron microscopy
of differentiating chick ovaries during
embryonic period
Murakami, M., I. Iuchi and K. Yamagami:
Isolation of intact yolk spheres of fish
embryos, which contain the majority of lyso-
somal acid phosphatase responsible for yolk
Nakamura, S., R. Kagotani, H. Fujisaki and
M. K. Kojima: The acid-insoluble organic
matrix of spicules in the sea urchin
Hemicentrotus pulcherrimus .........-..--. 741
Ohya, Y., K. Watanabe, N. Shimamoto and
M. Amano: Scleral fibroblasts of the chick
embryo can proliferate without transferrin in
protein-free culture
Inoue, C. and Y. Kakinuma: Symbiosis be-
tween Cytaeis sp. (Hydrozoa) and Niotha
livescens (Gastropoda) starts during their
larval stage
Amemiya, S. and Y. Nakajima: First electron
microscopical study on the sperm morpholo-
gy of the sea lily (Crinoidea, Echinodermata)
(COMMUNICATION)
Endocrinology
Kobayashi, M., M. Amano, Y. Hasegawa, K.
Okuzawa and K. Aida: Effects of olfactory
tract section on brain GnRH distribution,
plasma gonadotropin levels, and gonadal
stage in goldfish
Madsen, S. S. and H. A. Bern: Antagonism
of prolactin and growth hormone: Impact on
seawater adaptation in two salmonids, Salmo
trutta and Oncorhynchus mykiss
Kai-ya, H., J. Okuyama, T. Ishijima, Y.
Sasayama, H. Yoshizawa and C. Oguro:
Effects of Ca concentrations in culture
medium on the release of calcitonin from
incubated ultimobranchial glands of the bull-
frog, Rana catesbeiana
Kobayashi, Y., S. Kawashima, S. Takahashi
and K. Wakabayashi: Effects of chronic
phosphoprotein metabolism (COM- treatment with chlorpromazine on the aging
MUNICATION)? = 2seetaseeeeaeene ene 891
(Contents continued on inside back cover)
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October 1992
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LOOLOGICAL SCIENCE
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ZOOLOGICAL SCIENCE 9: 901-924 (1992)
REVIEW
Heterogeneity and Development of Somatotrophs and
Mammotrophs in the Rat
SuMIO TAKAHASHI en
Department of Biology, Faculty of Science, Okayama University,
Tsushima, Okayama 700, Japan
ABSTRACT— Growth hormone (GH) and prolactin (PRL) are secreted from corresponding secretory
cells, somatotrophs (GH cells) and mammotrophs (PRL cells) in the pituitary gland. A novel cell
type, the mammosomatotroph (MS cell), which contains both GH and PRL in the same cell, has been
found in many species including rats, mice and humans. In this article, we demonstrate the
heterogeneity of GH cells and PRL cells at various levels, mainly in the rat, and discuss the
developmental and functional significance of these heterogeneities. Age-related alteration in GH and
PRL secretion is summarized, since it is one of the most important aspects of the developmental
changes in pituitary glands. Heterogeneities of GH and PRL cells may be the outcome of various
difference in the molecular variance of hormones, the intracellular age of hormones, the difference in
receptors on the pituitary cells for hypothalamic regulatory hormones, the different mechanisms of
intracellular signal transduction, and the location of pituitary cells in the gland, and may also reflect
the maturation of pituitary cells. The possible relationship among GH cell, PRL cell and MS cells is
© 1992 Zoological Society of Japan
discussed.
INTRODUCTION
Growth hormone (GH) and prolactin (PRL) are
protein hormones that are produced in corre-
sponding specific pituitary secretory cells, somato-
trophs (GH cells) and mammotrophs (PRL cells)
[1]. A significant amount of evidence has indicated
that each cell population of GH cells and PRL cells
consists of morphologically and functionally heter-
ogeneous cells [2, 3]. Furthermore, variants of
hormone molecules have been found. Such heter-
ogeneity observed may reflect the functional dif-
ference or maturating process of pituitary cells and
hormone molecules.
A novel pituitary cell, a mammosomatotroph
(MS cell) or a somatomammotroph, which con-
tained GH and PRL in the same cell, was found in
several species. In this paper, the term mammoso-
matotroph is used to describe this secretory cell.
The developmental and functional significance of
Received July 10, 1992
MS cells is not yet fully understood. It is notewor-
thy that GH and PRL genes are believed to be
derived from a common ancestral gene [4, 5], and
are regulated by a common transcription factor [6,
7]. Accordingly, MS cells are considered to be
closely related to GH cells and PRL cells. Im-
muno-electron-microscopical studies showed sub-
types of GH cells and PRL cells in rat pituitaries.
MS cells in the rat closely resembled one of the
subtypes of GH cells and PRL cells. This finding
suggests that MS cells may be involved in the
genesis of GH cells and PRL cells. In this paper,
the morphological and functional heterogeneity of
GH cells and PRL cells, and the relationship
among the three pituitary cells, GH, PRL and MS
cells, will be discussed using examples mainly from
the rat. As one of the most important develop-
mental aspects in pituitary glands, age-related al-
terations in GH and PRL secretion will be exten-
sively studied in order to understand the signif-
icance of the heterogeneities of GH and PRL cells.
The molecular heterogeneity of both hormones
902 S. TAKAHASHI
will also be briefly considered.
I. SOMATOTROPHS
1. Identification of GH cells
Size, shape and distribution Immunocytochem-
ically identified GH cells in rat pituitaries ranged
from ovoid to pyramidal in shape, and were usual-
ly situated along sinusoids. They were evenly
dispersed bilaterally and rostrocaudally, but un-
evenly distributed dorsoventrally [8]. GH cells
were not found near the intermediate lobe, nor
were they found in the anteroventral portion of the
gland [9, 10].
Differentiation of GH cells The ontogeny of
GH cells has been immunocytochemically studied
in several laboratories. GH cells were first
observed at 18 or 19 days of gestation in the rat
(the day on which the vaginal plug was detected is
designated as day 0 of gestation) [11-13]. Using a
combination of the immunocytochemistry and the
in situ hybridization method [14], GH im-
munoreactivity was detected from day 18 of gesta-
tion, but GH-mRNA positive cells were detected
on day 19 of gestation. GH cells had substantially
increased in number by day 19 of gestation. Pitui-
tary specific transcription factor GHF-1 (Pit-1),
which was responsible for activating GH and PRL
genes, was detected in the anterior pituitaries on
day 16 of gestation [15], although GH-mRNA and
GH immunoreactivity were first expressed on day
18-19 of gestation as stated above. In mouse
pituitaries, temporal and spatial correlation be-
tween the GHF-1 gene expression and GH gene
expression was clearly observed [16]. GHF-1
transcript was first detected on day 13 of gestation,
and had significantly increased by day 15 of gesta-
tion. GH-mRNA was first detected on day 15 of
gestation. GH synthesis increased sharply be-
tween the 16th and 17th day of gestation [17]. GH
cells were morphologically detected on day 16 of
gestation [16].
2. Morphological heterogeneity of GH cells
Immuno-electron-microscopical studies have
shown that there are three subtypes of GH cells in
the rat [18]. GH cells were classified based on the
size of secretory granules (Fig. 1). The Type I GH
cell contained large secretory granules (diameter,
250-350 nm, Fig. 2). The Type II GH cell con-
tained large and small secretory granules (di-
ameter, 100-150 nm, Fig. 3). The Type II] GH
cell contained small secretory granules (Fig. 4).
Type I and II cells are polygonal or oval in shape,
and medium in cell size. Type III cells are oval or
sometimes irregular in shape, and small in size.
The rough endoplasmic reticulum and the Golgi
apparatus were moderately developed in Type I
and II cells. Type I and II GH cells had been
originally identified as somatotrophs. The relative
proportion of each subtype of GH cells differed
between male and female rats (Fig.5), and
changed with several hormonal treatments and age
[19]. This will be discussed later.
3. Development of GH cells
Proliferation of pituitary cells The pituitary
gland is an “expanding organ” (see Goss, [20]),
and its development is characterized by the fact
that cell differentiation is not incompatible with
mitosis, and every pituitary cell probably has the
capacity to divide during its life span [21]. Mitotic
GH cells were immunocytochemically identified in
several reports [22-25]. These observations clear-
ly indicate that differentiated GH cells divide in a
similar manner to other types of pituitary cells [26-
30]. Pituitary cells proliferate using a mode of
self-duplication, and the differentiated pituitary
cells divide into the same two differentiated cells.
However, another mode of proliferation cannot be
ruled out. For example, undifferentiated “stem”
cells, may divide and then one of the divided cells
may terminally differentiate into a specific secre-
tory cell.
Sexual difference in the percentage of GH cells
A sexual difference in the number of GH cells was
found in rats [19, 31] and in mice [32]. Our study
showed the percentage of GH cells in male and
female rats at various ages (Table). The percent-
age of GH cells was higher in male rats than that
in female rats, and decreased with age in both
sexes. Similar findings were reported by a reverse
hemolytic plaque assay [33]. On the contrary,
Dada er al. [34] could not detect the sexual differ-
ence in the percentage of GH cells, although the
GH and PRL Cells in the Rat 903
Fic. 1. Secretory granules immunocytochemically stained with rat GH antiserum by the protein A gold colloid
method. Large secretory granules with a diameter of 250-350 nm (arrows) and small secretory granules with a
diameter of 100-150 nm (arrowheads) are seen. Bar: 200nm. (from Takahashi [19]).
Fic. 2. Type I GH cell in a 6-month-old female rat. Large secretory granules are seen throughout the cytoplasm.
Bar: 1.0 4m. (from Takahashi [19]).
Fic. 3. Type Il GH cell in a 6-month-old female rat. Large and small secretory granules are seen throughout the
cytoplasm. Bar: 1.0 zm. (from Takahashi [19]).
Fic. 4. Type III GH cell in a 6-month-old female rat. Small secretory granules are seen throughout the cytoplasm.
Bar: 1.0m. (from Takahashi [19]).
reason for this discrepancy is not clear. proliferation of GH cells [35]. Thus, hypothalamic
Hormonal effects on GH cells Growth hor- __ peptides affect the proliferation of pituitary cells
mone-releasing hormone (GHRH) stimulates the _[30, 36]. GHF-1, a transcription factor of the GH
904
Fic. 5.
S. TAKAHASHI
CJ Type | Type Il
Male
3 an
) °
fo} ° ox
sy
to}
20
Percentage of GH cell types
Female
Percentage of GHcell types
o
to}
Age
Age-related changes in the percentages of GH cell types in male and female rats. The number above the
GB Type 1!
(months)
columns depicts the number of rats. Bars depict the standard errors of means. The percentage of Type I cell
increased, whereas the percentages of Type II and III cells decreased at 2 and 6 months of age. At 12 and 18
months of ages, the percentages of Type II and III cells increased. (from Takahashi [19]).
TABLE
Percentage of GH cells and PRL cells in
male and female rats
Percentage of cells (%)”
Sex Age(months)
GH cell PRL cell
Male 68.34+2.2)" 15.5+1.4
12 59.7+4.1° 22.1+1.6
18 40.5+4.2" 18.9+3.0
Female 40.6+2.0° 34.0+4.4%
12 34.8+4.1° 48.7+2.9"
18 PB NAA Spies (le
') Mean+S.E.
>) In each age-group five rats were used for the
determination of the percentages of GH cells and
PRL cells.
Statistical significance was tested by ANOVA.
When significant, the differences among age-groups
of each sex were determined by Duncan’s multiple
range
test. In each sex, there is a_ significant
difference between the values for the age-groups
with the same superscripts. a, P<0.05; b, c,d, P<
0.01.
(from Takahashi er al. [31]).
gene, may function in the proliferation of GH cells
[37]. This suggests that stimulation of GH gene
expression may stimulate the cell division of GH
cells.
Estrogen decreases the percentage of GH cells,
but androgen increases it [38, 39]. Estrogen in-
creased the percentages of Type II and III GH
cells (Fig. 6), and androgen increased the percen-
tage of Type I cells and decreased the percentage
of Type II cells (Fig. 7). As estrogen is known to
inhibit GH secretion, and androgen is known to
stimulate [39], it is concluded that the inhibitory
factors for GH secretion decrease the percentage
of GH cells, and the relative proportion of Type I
cells, and, on the contrary, the stimulatory factors
for GH secretion increase the percentage of GH
cells and the relative proportion of Type I GH
cells. T3 is known to stimulate fetal somatotroph
differentiation probably by a synergistic action
with cortisol [40] and GH production [41]. T
GH and PRL Cells in the Rat 905
» L_J]Type! KNtypel! Btype i
60
40
20
Percentage of GH cell types
(0)
Vehicle ES
Fic. 6. Effects of injection of 50 ug estradiol-172 (E>)
daily for 5 days on the percentage of GH cell types in
male rats. In E>-treated rats, the percentage of
Type I cells decreased and the percentage of Type II
and III cells increased. The number above the
column depicts the number of rats. Bars depict the
standard errors of means. *P<0.05, **P<0.01 vs
vehicle. (from Takahashi [19]).
treatment (10 ~g/100 g BW daily twice for 5 days)
significantly increased the percentage of Type III
GH cells from the control level of 3.4+1.1% (n=
6) to 6.9+0.8% (n=6). This increase in Type III
cells may indicate the formation of immature GH
cells (Type III) from undifferentiated cells, pro-
vided that the Type HII GH cell is an immature
type of GH cell. Thus, the relative proportion of
GH cell subtypes changed, together with changes
in GH secretion, indicating that three morphologi-
cally different GH cell types may have different
secretory activities.
Development of GH cells Perinatal develop-
ment of GH cell subtypes was reported by Kuro-
sumi and Tosaka [42], and postnatal changes (from
immature ages through to aged ones) were studied
by Takahashi [19]. The Type III GH cell was the
predominant type of GH cell during the prenatal
period, and the percentages of Type I and II cells
2 L ]typel KYtypen Bi typem
Kk
Vehicle
Fic. 7. Effects of injection of 100 ug testosterone prop-
ionate (TP) daily for 5 days on the percentages of
GH cell types in male rats. In TP-treated rats, the
percentage of Type I cells increased, but the per-
centage of Type II cells decreased. *P<0.05 vs
vehicle. (from Takahashi [19]).
gradually increased by the term. After birth, the
Type I GH cell became the predominant type.
Such morphological changes in GH cell popula-
tions have been extensively described [19]. Type I
cells, containing large secretory granules (250-350
nm in diameter) predominated throughout the life
(Fig. 5). The proportion of Type I cells was
highest at 6 months of age. The proportion of
Type Il and of Type III cells decreased from 1
month to 6 months of age, but increased there-
after. This suggests that when GH secretion is
more active (age-related changes in GH secretion
will be discussed later), the proportion of Type I
cell increases, and when GH secretion is less
active, the proportion of Type II and III cells
increases. GH cell-populations, morphologically
classified, changed in accordance with GH secre-
tory activity. Thus, it is highly probable that
morphological heterogeneity of GH cells reflects
906 S. TAKAHASHI
functional heterogeneity and/or the maturating
process of GH cells. Therefore, as originally
stated by Kurosumi er al. [18], the Type III GH cell
may be an immature type of GH cell, the Type I
cell the mature type, and the Type II cell may be
an intermediate type, although no direct evidence
for this hypothesis has been presented.
The total volume of each GH cell type had been
estimated [19] (Fig. 8), although the number of
each type of GH cell was not examined. Type I
cell populations peaked in volume at 6 months of
age, and decreased thereafter. Type II and III cell
populations increased in volume with age. The
change in volume of GH cell populations may be
partly due to the changes of the number of GH
cells. Type I GH cells may increase with age until
at least 6 months of age. Type II and III cells may
gradually increase with age (Fig.8). These
changes in percentages and estimated number of
each GH cell type may be explained in several
ways. One is that the increase in the percentage
and number of GH cells is caused either by the
proliferation of a specific type of GH cells, and/or
the cell death of other specific types of GH cells.
Another way is the conversion of one type of GH
Male Female
3 Type I
2
2
=i
oO
3c
eS ©
0 Type Il
© 1
aot TT
3
© oO
>
1 Type Ill
)
1 2 61218 1 2 6 1218
Age (months)
Fic. 8. Estimated total volumes of each GH cell type.
Volumes were expressed in arbitrary units. Age-
related differences were detected in each GH cell
type of both sexes (male, for each cell type: P< 0.01;
female, Type I: P<0.01, Type II, Ill: P<0.05).
(from Takahashi [19]).
cell to another type of GH cell. It is also possible
that GH cells are generated from stem cells or
progenitor cells, although their presence has still
not been proved.
The interconversion of one type of GH cell to
another type of GH cell is the most probable
among the three possibilities. We have not direct
evidence for it, but if large secretory granules are
formed as a result of more intense stimulation
from the hypothalamus (probably GHRH), the
new formation of large secretory granules in Type
III cells is to convert Type III cells to Type II cells.
In Type II cells the formation of the small secre-
tory granules may gradually slow or stop, and the
proportion of the small granules becomes even-
tually smaller. As the result, the mature Type I
cells will finally appear. Alternatively, the small
secretory granules may be fused to be a large
secretory granule as previously reported in PRL
cells [43]. For another example, estrogen in-
creased the percentage and number of Type II GH
cells (Fig. 6), and it had already been verified that
the proliferation of GH cells is not stimulated by
such estrogen treatment [44]. Therefore, estrogen
appears to cause the conversion of Type I cells to
Type II cells. The small secretory granules may be
newly formed in the Golgi apparatus, or the large
secretory granules may be disintegrated to the
small granules under estrogen treatment. The
transition from the Type I cell to the Type II cell is
more probable than the proliferation or genesis of
the Type II cell.
4. Functional heterogeneity of GH cells
Heterogeneity in GH synthesis Uptake of [*H]-
leucine into dissociated GH cells was studied using
electron microscopic autoradiography. Only half
of the GH cells were heavily labelled, indicating
that GH synthetic ability differed among GH cell
populations [45]. Furthermore, dissociated pitui-
tary cells were separated into two subpopulations
of GH cells by density gradient centrifugation.
One of the two GH-cell subpopulations, which is
less dense (the light fraction), produce more GH
than the other [46].
Heterogeneity in GH release GH release from
dissociated individual GH cells was analyzed by a
reverse hemolytic plaque assay. Figure 9 shows
GH and PRL Cells in the Rat
30
nN
a
—
nN
oO
Frequency (% of total plaques)
° a
on
ee
a
\\7
\4 \.
ee ee, eee 0
le} 1 2 345 10 20 40 80 160
Plaque area (um’x 10°)
Fic. 9. The composite distribution pattern of plaque
areas formed by GH cells from four hemolytic
plaque assays. Each points are means of 4 separate
assays with the standard errors. In each assay about
three hundred plaques were measured.
the bimodal frequency pattern of the plaque areas
formed by GH cells of adult female rats. One
subpopulation formed the larger hemolytic pla-
ques than the other. As the plaque area is pro-
portional to the amount of hormones secreted, this
result indicates that one group of GH cells secretes
more GH than the other. Similar result has been
already reported by Frawley and Neill [47],
although the bimodal distribution of the plaque
area was detected only in GHRH-treated pituita-
ties. The subpopulation of Type III GH cells was
smaller than the other two subpopulations of Type
I and II cells. Consequently, contribution of Type
III GH cells in the reverse hemolytic plaque assay
is quite small, and can be neglected. In young
female pituitaries, the relative proportion of Type
II cell-populations to Type I cell-populations was
41%. It is probable that the two subpopulation,
morphologically divided, may correspond to the
two subpopulation detected in the reverse hemoly-
tic plaque assay. Further study is needed to clarify
this correlation.
5. Possible mechanisms of functional heteroge-
neity
Preferential release of newly-synthesized hor-
mones Chen et al. [48] found, using the reverse
907
hemolytic plaque assay, that in basal secretion of
GH, one subpopulation of GH cells secreted a
larger amount of GH than the other. The former
subpopulation was likely to preferentially release
newly-synthesized hormones for basal secretion,
whereas the other population was likely to release
stored hormones for basal secretion, even without
stimulation. Therefore, this heterogeneity of GH
cell population may be due to the difference in the
intracellular content of the preferentially releas-
able hormone component among GH cell popula-
tions.
Difference in GH cell-location A tissue-slicing
method clarified another functional heterogeneity
of GH cells [8]. The responsiveness of GH cells to
GHRH on GH release is different, depending on
the location of GH cells within the gland. GHRH-
induced GH release was only detected in GH cells
derived from the left dorsorostral, right ventro-
caudal and right ventrorostral parts of the gland.
This location-dependent functional heterogeneity
of GH cells may be due to the differential blood
supply, the different concentrations of hypothala-
mic hormones in different portal vessels, or the
effect of cell-to-cell communication (the paracrine
effect).
Differences in intracellular signal transduction
Localization of protein kinase-C (PK-C) subtypes
in the pituitary gland was studied immunocytoche-
mically [49]. Not all of the pituitary cells contained
PK-C. As for GH cells, only 9% of all GH cells
contained PK-C. This finding suggests that GH
cell populations can be divided into two sub-
populations by the difference in the PK-C system.
The function of PK-C in pituitary glands is not
fully understood, although numerous physiological
functions of PK-C are known [50]. As PK-C plays
a key part in intracellular signal transduction, the
GH cells which are deficient in PK-C may be
controlled by other signal transduction mecha-
nisms, Or may not have some functions (for exam-
ple, as PK-C is known to be involved in cell
proliferation in some types of cells, PK-C deficient
cells may not have the ability to proliferate).
6. Molecular heterogeneity of GH
Multiple molecular forms of GH are found in
the rat pituitary gland [51-54]. Farrington and
908 S. TAKAHASHI
Hymer [54], for example, demonstrated that 11
variants of rat GH exist, ranging in molecular
weight from 11 kDa to 88 kDa (Fig. 10). Some of
them were dimeric and glycosylated (24 K, Bollen-
gier et al., [53]). These variants might be related to
the functional heterogeneity of GH hormones, and
the morphological heterogeneity of secretory gra-
nules. Multiple forms of GH molecules were also
well known in human, bovine and ovine pituitaries
[S55, 56]. The most extensive studies were carried
out on human GH variants. Human pituitary
extracts contained at least half a dozen peptides.
The major GH components in pituitary extract are
20 kDa, 22 kDa (the major component), and 45
kDa (dimer) [57, 58]. Using transgenic mice. 22
kDa, 20kDa and 5 kDa variants of human GH
were evaluated [59]. Both 22 kDa and 20kDa
forms stimulated linear body growth and liver
hypertrophy. The linear growth mediated by the
22 kDa variant did not correlate with an increase
in blood IGF-1 level. The 5 kDa variant did not
Absorbance
o o
+++
| B
at
J ow
i o+
x at
é
ar
5 OT
—]
80-88K > 5 i
o wn
60-66K ee
E) Oar
, 3
geen: Bs See
40-44K = ie a
# N
sagt) = a o
25k > | 2a = ;
22K ot 22K of
20K ‘ = 20K
14K > = 14K <=
! as
s
Fic. 10. Representative Western blots of rat pituitary
GH contained in extracts electrophoresed under
nonreducing (A) or reducing (B) conditions (see,
Farrington and Hymer [54]). The reflectance optical
density tracking of blot in A is shown in the middle.
(from Farrington, M. and Hymer, W. C., Growth
hormone aggregates in the rat adenohypophysis,
Endocrinology, 126: 1630-1638, 1990; © The Endo-
crine Society, with written permission).
elicit any obvious activity. Thus, molecular
variants of hormones may have different functions,
although it has still not been established whether
each molecular variant has a physiological function
[S58]. Further studies on the mechanism of dif-
ferential production of each variant and on cellular
localization of each variant will probably give us
the answer to this issue.
7. Age-related changes in GH secretion
Physiological significance Multiple physiolo-
gical roles of GH have been reported previously
[60]. GH is one of the most important anabolic
hormones. Dysfunction of the GH _ secretory
mechanism may cause severe anomalies in various
body functions. Sonntag ef al. [61] found a de-
crease in protein synthesis, and GH administration
reversed this. Takahashi and Meites [62] also
reported the alterations in liver GH receptors with
age, and GH administration in old rats reversed
the age-related changes. The decreased GH secre-
tion resulted in a low plasma somatomedin-C level
[62]. Therefore, it is important to study the
age-related changes in GH secretion.
Changes in morphology of GH cells with age
Morphological changes of GH cells have already
been explained in the section Development of GH
cells. DNA content of GH cell-populations, which
indicate the number of GH cells, was estimated
from pituitary DNA content data and the percen-
tage of GH cells. The estimated DNA content
constituting the GH-cell population did not change
at 6, 12 and 18 months of age in male rats (16.1+
2.4, 18.9+2.1 and 13.9+1.8 ug, respectively), but
increased during this period in female rats (11.3+
1.6, 18.4+2.1 and 18.1+0.4 ug, respectively).
Changes in GH release with age GH is released
in a pulsatile fashion. In male rats, the pulse
interval is 3-4 hours, and in female rats it is about
70 minutes [63, 64]. The pulse amplitude is higher
in male rats than in female rats. GH secretory
patterns in male rats continued to remain un-
changed throughout the day and night, but
swiched to a rapid, highly pulsatile pattern at night
in female rats [65]. There was no clear relationship
between the GH secretory pattern and the phase
of estrous cycle [65]. These sexual differences in
GH secretion are clearly discussed in the review by
GH and PRL Cells in the Rat 909
ny
fo}
oO
Plasma GH (ng/ml)
~<
°
Cc
=}
a
11 12 13 14 15 16 11
Fic. 11.
Middle-aged
12 13 14 15 16 11
Clock time
Mean plasma GH concentrations in young (5 months), middle-aged (11 months) and old (25-29 months)
Old
12 ie 4 WS We
female rats of SD strain. Each point represents the mean of GH levels in 17 young, 6 middle-aged and 13 old rats.
Bars depict the standard errors of means. Plasma GH levels in young females were higher than in middle-aged
and old female rats. Plasma GH levels in old rats tended to be somewhat lower than in middle-aged rats. (from
Takahashi et al. [68]).
Jansson et al. [66]. In the rat, GH secretion
diminished in both sexes with age [67, 68] (Fig.
11). The pulse intervals did not change with age,
but the pulse amplitudes were significantly lower in
old rats than in young rats. GHRH responsiveness
to GH release was reduced in old rats in vivo [69,
70], although Weherenberg et al. [71] reported the
opposite result that there were no age-related
changes in the responsiveness to GHRH. A recent
study showed, using a hemolytic plaque assay, that
GH release from individual GH cells was less in
old female rats compared to young females (Taka-
hashi, unpublished observation). The mean pla-
que area produced by GH cells was significantly
lower in 20-21 month-old females (7.7 +2.6 x 10°
ym?) than that in 3-4 month old young females
(14.9+2.4x 10° wm’). Responsiveness to GHRH
was reduced in old female rats than in young rats.
Accordingly, the diminished GH secretion in old
rats is due partly to the reduced secretion of GH
from individual GH cells.
Changes in GH synthesis with age Pituitary GH
content decreased with aging [19]. GH synthesis in
young, middle-aged and old rats was studied at the
GH-mRNA level [31]. GH concentration per
single GH cell was significantly lower in old rats
than in young rats (Fig. 12). Also, GH-mRNA
concentration per single GH cell was significantly
lower in old female rats (Fig. 13). Thus, GH
synthesis diminished with age at the transcription
level of the GH gene. These results were in good
agreement with a recent in situ hybridization study
[72]. This decrease may be due partly to the
reduced release of hypothalamic GHRH in old rats
[73-74], and the reduced binding sites of GHRH
in old rats [75]. An uncoupling between the
Male Female
10007 100
(2) GH content
800 8 = GH concentration 80
600 60
400 * 40
@
(WNC [199-H® 371/377)
uonemuaou09 HO
200 tes Kk 20
GH_ content (wg/gland)
a
fo}
6 12 18 6 12 18
C) prt content 8 RK
200 GB PRL concentration KK
15
10
PRL content (wg/gland)
(VN 1199-Tud 371/317)
uoenusouos 7 yd
(1)
(o}
6 12 18 6 12 18
Age (month)
Fic. 12. Pituitary GH and PRL content (per gland) and
concentration (per ~g GH-cell DNA or PRL-cell
DNA) in male and female rats at 6, 12 and 18 month
of age. Female rats at estrus or persistent estrus
(middle-aged and old) were used. *P<0.05, **P<
0.01 compared with 6-month-old rats. *P<0.05,
**xP<0.01 compared with 12-month-old rats.
(from Takahashi et al. [31]).
910 S. TAKAHASHI
*lo Male Female
120
100
mo)
&
= 80
Z
w~ 60
=
=
5 40
20
7 7
to)
6 12 18 6 12 18
°lo
120
Z
A 100
8 80
a0)
Os *
oD
=
s 40
fod
— 20
<=
= i i 7
6 12 18 6 12 18
Age (month)
Fic. 13. Total pituitary GH mRNA content (per gland)
and concentration (per ~g GH-cell DNA) in male
and female rats at the age of 6, 12 and 18 months.
Female rats at estrus or persistent estrus (12 and 18
months) were used. *P<0.05 compared with 6-
month-old rats. (from Takahashi ef al. [31]).
GHRH receptor and the G protein occurred in old
male rats, resulting in a weaker response to
GHRH in old pituitaries [76]. In male mice GH
mRNA levels decreased with age [77].
Il. MAMMOTROPHS
1. Identification of PRL cells
Size, shape and distribution PRL cells were
found sparsely in the anterior-ventral portion of
the gland, and found in the areas near the in-
termediate lobe in the rat [9, 10, 78]. Regional
distribution of PRL cells was reported by Sasaki
and Iwama [79] in mice. The densities of PRL cells
in the rostral and caudal pituitaries of mice were
significantly greater than those of GH cells. The
number and size of PRL cells differed significantly
between male and female rats. PRL cells were
polygonal, elongated and frequently cup-shaped
and surrounded by large oval gonadotrophic cells
[80].
Differentiation of PRL cells There are several
reports about the first appearance of PRL cells in
rat pituitaries, and these are somewhat contradic-
tory. Sétal6 and Nakane [11] found PRL cells on
day 16 of gestation. Chatelain ef al. [12] reported
that PRL cells were detected on day 21 of gesta-
tion, and Watanabe and Daikoku [13] reported
that PRL cells were first detected postnatally.
Nogami et al. [14] observed immunocytochemical-
ly and by in situ hybridization that PRL was
detected on day 18-19 of gestation, and PRL-
mRNA was also detected on day 18-19 of gesta-
tion. PRL gene expression during the neonatal
period had been studied [81]. PRL genes were
expressed by at least 3 days of age, but the
translation of the PRL message was, interestingly,
reported to be blocked by the lack of association of
the PRL message with ribosomes. In the mouse
pituitary, immunoreactive PRL cells were detected
at birth, but PRL cells might possibly appear in
fetal pituitary glands [82]. In mice pituitaries an in
situ hybridization with a PRL riboprobe, actually
showed a few PRL cells 15.5 days after conception,
and the number of PRL cells decreased 16.5 and
17.5 days after conception, although GH cells
remarkably increased in number during this period
[16]. In another study, PRL synthesis in mice was
first detected at 8 days of age by a two-dimensional
Fic. 14. Type I PRL cell in an adult female rat. The cell is elongated and a round nucleus is located slightly
eccentrically. The rough endoplasmic reticulum and the Golgi apparatus are well developed. Large round or
irregularly shaped secretory granules are located in the peripheral cytoplasm. Bar=1.0 um. (from Takahashi
and Miyatake [88]).
Fic. 15.
Type II PRL cell in an adult female rat. Type II cells contain round secretory granules with a diameter of
150-250 nm. The number of secretory granules is larger than that in Type I cells. Bar=1.0 ~m. (from Takahashi
and Miyatake [88]).
at
sin the R
and PRL Cell
GH
912 S. TAKAHASHI
electrophoresis [17]. This discrepancy is partly due
to the difference in the sensitivity of assays used (in
situ. hybridization, immunocytochemistry, and
two-dimensional electrophoresis).
2. Morphological heterogeneity of PRL cells
Sato [80] studied postnatal development of PRL
cells in the rat, and suggested from an immunocy-
tochemical study that the oval PRL cells were
premature, the polygonal ones mature, and the
cup-shaped ones particularly differentiated. The
ultrastructure of PRL cells had been extensively
studied and three types of PRL cells, mainly based
on the size of the secretory granules, were found
[83-85]. Smets er al. [78] subdivided rat PRL cells
into two types, one containing large polymorphic
granules, and the other small round granules.
Harigaya et al. [86] also classified mouse PRL cells
into three types by immuno-electron-microscopy.
Electron microscopically, the PRL mRNA was
localized in the rat pituitary, and two types of
PRL-synthesizing cells were identified [87]. One
type was characterized by large secretory granules,
and the other by small secretory granules. Taka-
hashi and Miyatake [88] observed three subtypes
of PRL cells in the rat, and classified them based
on Kurosumi’s classification (Kurosumi et al. [89]).
Type I cells contained irregularly shaped large
secretory granules with a diameter of 300-700 nm
(Fig. 14). Type II cells contained spherical gra-
nules with a diameter of 150-250 nm (Fig. 15).
Type III cell contained small round granules with a
diameter of 100 nm (Fig. 16). Type I PRL cells
had been originally identified as mammotrophs.
3. Development of PRL cells
Sex difference in PRL cells Sex differences in
PRL cell number were immunocytochemically
found in mice [32] and in rats [90]. On the
contrary, Dada et al. [34] reported that sex differ-
Fic. 16. Type III PRL cell in an adult female rat. The cell is characterized by the small amount of cytoplasm
containing small secretory granules with a diameter of about 100 nm. Cell organelles are less developed. Bar=
1.0 um. (from Takahashi and Miyatake [88]).
GH and PRL Cells in the Rat 913
ences were not detected in adult rats. Using the
reverse hemolytic plaque assay, postnatal develop-
ment of PRL cells was studied, and it was clearly
shown that the percentage of PRL-secreting cells
did not differ between male and female rats at
immature ages, but significantly increased in adult
female rats as previously reported [33, 91]. These
sex differences in the percentage and the number
of PRL cells were caused by the difference in
estrogen level [90, 92-94].
Proliferation of PRL cells The proliferation of
PRL cells is stimulated by estrogen [29], and is
closely correlated with PRL secretion. Bromocrip-
tine, a dopamine agonist, inhibited not only PRL
%
1007 () Wee!
Type Il
BB ype ill
80
”
o
roe
=
> 60
(6)
—l
ioe
a
i)
oD
& 40
Cc
o
=
o
ra
20
(o)
Control Estradiol
Fic. 17. Effects of injection of 50 4g estradiol-17 (E>)
daily for 5 days on the percentage of PRL cell types
in male rats. In E;-treated rats, the percentage of
Type I cells increased and the percentage of Type II
and III cells decreased. The number above the
column depicts the number of rats. Bars depict the
standard errors of means. Estrogen affected the
relative proportion of each subtype of PRL cells (P
<0.01). (from Takahashi and Miyatake [88]).
secretion, but also the mitosis of PRL cells [44,
95]. The difference in the number of PRL cells
may partly result from the difference in the mitotic
activity of PRL cells. The sex difference in the
number of PRL cells is explained by the difference
in the mitotic activity of PRL cell. Actually, the
mitotic activity of PRL cells in estrous female rats
was significantly higher than that in male rats [29].
Development of PRL cells Estrogen increased
the percentage of Type I PRL cells, and decreased
the percentages of Type II and III cells (Fig. 17).
On the contrary, ovariectomy and bromocriptine
decreased the percentage of Type I PRL cells and
increased the percentages of the other two types
(Fig. 18). Thus, the relative proportion of PRL
cells changed in accordance with the change in
%
eS L_] Type!
NS Type I
= Type Ill
80
”
o
Q
or
5 OO
a
ra
ao
i)
o
ro)
S 40
c
®
2
o
a
20
(0)
Control
Bromocryptine
Fic. 18. Effects of bromocriptine treatment on the per-
centages of PRL cell types in adult female rats. The
number above the column depicts the number of
rats. Bars depict the standard errors of means.
Bromocriptine affected the relative proportion of
each subtype of PRL cells (P<0.01). (from Taka-
hashi and Miyatake [88]).
914 S. TAKAHASHI
PRL secretion. The Type III PRL cell, containing
small secretory granules, was predominantly pre-
sent at immature ages (Fig. 19), and was small in
size. Therefore, the Type III PRL cell is consid-
ered to be an immature type of PRL cell. Type I
PRL cell, containing large
secretory granules, constitutes most of the PRL
cell population in adult female rats and is large in
size. Thus, the Type I PRL cell is considered to be
a mature type of PRL cell. The Type II PRL cell is
considered to be an intermediate cell between the
Type I and III cell [88, 89]. The change of
secretory granules in size and shape is explained by
the fusion and lysosomal degradation of preexist-
ing secretory granules, which had previously been
shown by Farquhar et al. [43].
irregularly-shaped
100 (-] Type! Type | [EB Type tll
a
=> 80
3 5
—| 60
a
ro N
a 40 N
s N
= N
% 20 N
2 Ne
oa N
M F F M F
10 30 60
Age (days)
Fic. 19. Postnatal development of the percentages of
PRL cell types in male (M) and female (F) rats. The
number above the columns depicts the number of
rats. Bars depict the standard errors of means.
There are significant differences (P<0.01) between
these groups: 10-day-old males vs. 30-day-old males,
10-day-old females vs. 30-day-old females, 30-day-
old females vs. 60-day-old females. (from Taka-
hashi and Miyatake [88]).
4. Functional heterogeneity of PRL cells
Heterogeneity in PRL synthesis and release
Hymer et al. [96] separated PRL cell populations
using the differences in unit gravity, that is, the
difference in the cell shape and secretory granule
content. This method revealed that the intracellu-
lar content of PRL differed among the separated
PRL cell fractions, and the amount of PRL re-
leased during the culture period of 14 days was
positively correlated with the initial intracellular
PRL content [97]. Swearingen [98] first found the
heterogeneity in turnover of PRL in in vivo and in
vitro studies. Walker and Farquhar [99] further
clarified heterogeneity in PRL cells with respect to
the PRL synthetic rate, which was autoradio-
graphically visualized using the difference in the
uptake of [*H]-leucine in PRL cells. They also
found a subpopulation of PRL cells which secreted
preferentialy newly synthesized PRL. Velkeniers
et al. [100] separated PRL cell populations into
high density and low density populations using the
discontinous Percoll gradient, and found that low
density PRL cells have a high basal secretory
activity and a higher PRL-mRNA content, and
high density PRL cells have a low basal secretory
activity and a lower PRL-mRNA content, but a
higher responsiveness to vasoactive intestinal
polypeptide.
Functional heterogeneity of rat PRL cells was
also shown by the reverse hemolytic plaque assay
[38, 101, 102]. The bimodal distribution of plaque
sizes indicated that the amount of hormones re-
leased from dissociated individual cells differed
among PRL cells [101]. PRL cells were heter-
ogeneous with respect to basal hormone secretion
and responsiveness to TRH. Thus, there
apparently seemed to be at least two subpopula-
tions of PRL cells.
Other evidence for the functional heterogeneity
of PRL cell populations was reported by Arita er
al. {103, 104] using the sequential cell immunoblot
assay. Their study reported that there is a heter-
ogeneity in PRL cell populations with respect to
dopamine and TRH.
Heterogeneity in PRL-cell surface antigen
Another morphological heterogeneity in anti-PRL
cell-surface immunoreactivity was shown in the rat
pituitary [105]. Only half of all PRL cells from
female rat pituitaries contained a cell-surface PRL
immunoreactivity. This finding implies the pre-
sence of PRL receptors on the cell surface, or
some of the released PRL is retained on the
surface of these cells. From this finding, PRL cell
populations may also be divided into at least two
subpopulations. However, it is not easy to corre-
late this heterogeneity of PRL cell populations to
the PRL cell types stated above.
GH and PRL Cells in the Rat 915
5. Possible mechanism of functional heterogeneity
of PRL cells
Difference in PRL cell-location
dependent functional heterogeneity in PRL cells
was shown by the reverse hemolytic plaque assay
[106], similar to the findings that have already been
described in GH cells. In this study, PRL cells
from the peripheral rim (outer zone) responded
greatly to TRH, but only moderately to dopamine.
PRL cells from the central region (inner zone)
were affected slightly by TRH, but were markedly
inhibited by dopamine. These regional differences
in pituitary cells may be derived from the regional
differences in the portal blood levels of hypothala-
mic releasing/inhibiting hormones. Another pos-
sibility is the paracrine effect on pituitary cells
from the neighboring cells.
Difference in the molecular variants secreted
Diethylstibestrol-induced prolactinomas consisted
of three different subpopulations of PRL cells
[102]. In their study by gravitational seidmenta-
tion, PRL cells were divided into large-, intermedi-
ate- and small-sized PRL cells, which differed in
their content and release of PRL. Large- and
intermediate-sized PRL cells contained typical
pleiomorphic secretory granules, but small-sized
PRL cells were sparsely granulated or agranular.
Small-sized PRL cell-populations contained uni-
que PRL variants, whose molecular weights were
10-14K Dalton. This study suggests that there
may be a relationship between the molecular
heterogeneity of PRL and the diversity of mor-
phology and function of PRL cells. Molecular
variants of PRL will be discussed later.
Difference in electrophysiological properties of
PRL cells and dopamine receptors on PRL
cells PRL cell populations were electrophys-
iologically divided into two subpopulations, which,
in turn, correspond to two groups separated by a
BSA density gradient separation, the light and
heavy groups [107]. Most of PRL cells of the light
fraction showed a type 1 response; dopamine
induced a hyperpolarization of the membrane
potential from the resting potential. The other
PRL cells of the heavy fraction mostly do not
respond to a dopamine (type 2 response), but
when the membrane potential has been depola-
A. location-
rized, dopamine induces a repolarization. The
expression of the two dopaminergic D2 receptors,
D24,5 and D2444, was studied and was found to be
different in these two PRL cell populations [108].
The ratio D24;5/D2444 was higher in the light
fraction of PRL cells than in the heavy fraction.
This result indicates that the two different re-
sponses to dopamine in PRL cells could be associ-
ated with the differnetial expression of two differ-
ent D2 receptors. Such differences may eventually
bring about a difference in PRL secretion, and/or
even in the morphology of PRL cells.
Differences in the intracellular age of PRL The
intracellular age of PRL molecules in the pituitary
cells may be another important factor for the
functional heterogeneity of PRL cell populations
[109]. Dopamine had a significantly lower inhibi-
tory effect on mature PRL (4-8 hr after synthesis)
than newly synthesized and older stored PRL.
TRH had a greater stimulatory effect on mature
PRL (4-8 hr after synthesis), indicating that ma-
ture PRL molecules are more readily released than
on newly synthesized and old stored PRL. Thus,
functional heterogeneity in PRL cell populations
may be accounted for by the difference in the
intracellular age of PRL, which is determined by
whether it is newly synthesized or old (stored).
6. Molecular heterogeneity of PRL
Several laboratories described molecular
variants of PRL [110]. Hymer and Motter [102]
reported in diethylstilbestrol-induced prolactino-
mas that several variants of PRL molecules ranged
from 12 kDa to 64kDa. Bollengier et al. [53] also
showed molecular heterogeneity of PRL. That is,
23K, doublet 25K-26K, 40K and 42K. A
variant of 26 kDa is considered to be glycosylated
PRL. High molecular weight variants occur as a
product of disulfide linkage between monomeric
units. Oetting and Walker [111] reported an
interesting finding that three variants of PRL,
whose molecular weights were the same (24K),
were different in their net charge (isoform 1, least
negatively charged isoform of PRL; isoform 2;
isoform 3, most negatively charged isoform), and
considered them to be synthesized in PRL cells.
Isoform 2 was the predominant form inside the cell
and isoform 1 was the predominant secreted form,
916 S. TAKAHASHI
although all three isoforms were released.
Physiological significance of molecular hetero-
geneity Frawley et al. [112] indicated the possi-
bility that each molecular variant of PRL differs in
biological activity, and suggested that each
molecular form may have specific target cells, and
consequently, have specific physiological roles. A
good example to demonstrate the possible phys-
iological significance of molecular variants of PRL
was recently reported in ram pituitaries. The study
clearly showed that production of variant forms of
PRL in ram pituitary glands varied seasonably
[113]. In their study, the 23 K form is a primary
hormone, and the 25K form is a glycosylated
form. High molecular weight-forms (more than 25
K), which are aggregated by a disulfide linkage
between monomers, are significant in winter, and
may be for storage. During the season when PRL
secretion is active, high molecular forms dis-
appeared. An explanation for this could be that
synthesized hormones may be rapidly released into
circulation, and are not stored in the cell. On the
contrary, during the season when PRL secretion is
low or inhibited, synthesized hormones are more
likely to aggregate and to become the stored type.
Thus, it is probable that changes in molecular
forms of a hormone may be parallel to changes in
the secretory activities of hormones.
7. Age-related changes in PRL secretion
Physiological significance A number of phys-
iological actions of PRL have been reported [114].
The altered PRL secretion induces various dis-
eases (eg. [115, 116]). Therefore, it is valuable to
study age-related changes in PRL secretion.
Changes in morphology of PRL cells with age
Kawashima [117] reported, electron microscopi-
cally, the morphological changes in pituitary cells,
particularly hypertrophy and hyperplasia of PRL
cells in female rats, although an immunocyto-
chemical identification had not been done. Age-
related changes in immunocytochemically iden-
tified-PRL cells were reported by Takahashi and
Kawashima [90]. The percentage of PRL cells
significantly increased in female rats with age
(Table). The total number of PRL cells had not
been measured, but it had been estimated from the
pituitary DNA content and the percentage of PRL
cells as described in GH cells. Actually, the DNA
contents constituting the PRL-cell population at 6,
12 and 18 months were as follows; in male rats, 3.7
+0.6ug (7), 7.0+0.8 (8) and 6.5+0.8 (5), and in
female rats, 9.5+1.4 (8), 25.8+2.9 (8) and 40.3 +
2.4 (7). The DNA contents of PRL cells increased
in both sexes with age, but more markedly in old
female rats, suggesting the significant increase of
PRL cells in number. This was confirmed by
Chuknyiska ef al. [118]. The increase in the
number of PRL cells with aging was caused by the
Ovarian estrogen, the stimulatory factor for the
proliferation of PRL cells [119]. Prepubertal ovar-
iectomy prevented the increase of PRL cells in
number in old female rats.
Age-related changes in PRL cell mitosis in the
rat were observed (Fig. 20). Even in 2-year-old
female rats mitotic pituitary cells were encoun-
tered. Immuno-electron-microscopical studies
have been done in male rats [120]. The relative
proportion of each type changes with age in male
rats. One type of PRL cell, containing small round
secretory granules (Type III cells in Kurosumi’s
classification [89]), increased in percentage, and on
the contrary, another type of PRL cell with large
irregularly-shaped secretory granules (Type I cells
in Kurosumi’s elassification) decreased in percen-
tage in old male rats.
Changes in PRL synthesis and secretion with
age PRL secretion increased with age, and the
enhanced secretion of PRL is partly due to the
dysfunction of the hypothalamic dopaminergic
mechanism [121-123]. A reverse hemolytic plaque
assay revealed that the amount of PRL released
per cell decreased in old rats [124]. Pituitary PRL
content significantly increased with age in female
rats, but PRL concentration per PRL cell de-
creased [31] (Fig. 5). PRL mRNA levels per PRL
cell decreased with age in both sexes (Fig. 21).
PRL synthesis in each PRL cell decreased at the
transcription level with age. However, since PRL
cells significantly increased in number in old
female pituitaries of the Wistar/Tw rats [90], the
total amount of PRL significantly increased with
age. Stewart ef al. [125] recently reported no
significant change in PRL mRNA concentrations
(per measured amount of pituitary DNA) with age
in female rats, but did report a significant increase
GH and PRL Cells in the Rat 917
69.5%.
8
80 - (8)
73.7°lo
= () Male
E wall [2] Female
“ 25.2% positive cell
3 (8) 95.79,
: (6)
= 20) |e
=<
o
Uv
S
nl 65.3%
iS 201% NN 13.5% 15.5% a) 3 oe
= 11.9% 30.4% 25.0%
= (5) (6) (6) § (6
N (6) (7) (4)
E D2 E D2 E D2 PE PD
20 30 60 90 150 - 180 ca360 ca.730
Age (days)
Fic. 20. Age-related changes in the mitotic indices of pituitary cells and PRL cells in male and female rats. The
colchicine-arrested mitotic cells and immunocytochemically-identified PRL cells (positive cells) were observed.
For the detail of the method, see Takahashi er al. [29]. Female rats at estrus (E), 2nd day of diestrus (D3),
persistent estrus (PE) and persistent diestrus (PD) were used. The percentage above the each column depicts the
percentage of mitotic PRL cells in total mitotic pituitary cells. The number in parenthesis depicts the number of
rats, and bars depict the standard errors of means.
in serum PRL level. We did not find any signif-
icant difference in PRL mRNA concentrations
, Male Foner * (per yg of pituitary cell DNA) in female rats,
200 either (data not shown). Crew et al. [77] reported
2 an age-related decrease in PRL mRNA in male
> 150 mice.
<
Zz
=
5 100 Ill. MAMMOSOMATOTROPHS
&
eK
50 * 1. Identification of MS cells
: | z Mammosomatotrophs (MS cells), which con-
) De BR AB tained both GH and PRL in the same cell, were
ony! immuno-electron-microscopically described in in-
lo tact adult rats [126-128]. MS cells were small in
s 120 size and irregular in shape. Secretory granules,
F100 50-150 nm in diameter, contained both hormones
rs)
caieners [126].
x
Oo.
2 60 * nee
= ok Fic. 21. Total pituitary PRL mRNA content (per
s 40 ** gland) and concentration (per ng PRL-cell DNA) in
iz 7K male and female rats at the age of 6, 12 and 18
= 20 fe months. Female rats at estrus or persistent estrus
o 5 7 [al 7 (middle-aged and old) were used. *P<0.05, **P<
6 12 18 6 12 18 0.01 compared with 6-month-old rats. P<0.05
compared with 12-month-old rats. (from Takahashi
Age (month) et al. [31]).
918 S. TAKAHASHI
MS cells in various animals MS cells were quite
rare in normal adult rats, but MS cells were usually
encountered in lactating and pregnant females
[126, 127]. Adenomatous rat pituitaries contained
MS cells [128]. MS cells were also observed in
mice [32, 129], musk shrews [127], bats [130], cows
[131], sheep [132, 133], rhesus monkeys [134], and
humans (fetal, [135-137]; normal adult, [138];
adenomatous adult, [139-141]). MS cells in rat
pituitary tumor lines are well known [142-144].
However, Shirasawa et al. [145] could not detect
any MS cells in the fetal and male adult bovine
pituitary glands using three different immunohis-
tochemical methods. The difference between the
report of Fumagalli and Zanini [131] (nursing cows
and virgin cows) and that of Shirasawa et al. [145]
(fetal and adult bulls) is partly due to the differ-
ence in the age and sex of animals used. In mice,
MS cells were further divided into two subtypes,
the small, round, solid secretory granular type and
the vesicular secretory granular type [129].
Ishibashi and Shiino [127] found two types of
colocalization of GH and PRL. One type was that
GH and PRL are colocalized in the same secretory
granules within a single cell, as described by Niki-
tovitch-Winer et al. [126]. The other type is that
GH-secretory granules and PRL-secretory gra-
nules were intermixed within closely aggregated
and interdigitated cell-clusters which consist of GH
and PRL cells in pregnant rats and female musk
shrews. This type is similar to the multinucleated
mammosomatotrophs in cows reported by Fuma-
galli and Zanini [131]. This finding suggest a
possibility that the enhanced stimulation of hor-
mone secretion iduce the fusion of the secretory
cells.
2. Development of MS cells
Using the reverse hemolytic plaque assay, MS
cells were detected in neonatal and adult male and
female rat pituitaries [146], in bovine pituitaries
[147] and also in human pituitaries [135, 138].
Hoeffler et al. [33] reported that MS cells were
35.8% of all GH and/or PRL secreting cells of
5-day-old male rats. In adult male rats about one
third of all GH and/or PRL secreting cells are MS
cells [146]. Leong er al. [148] reported that about
5% of all pituitary cells were MS cells in adult male
Pues
Fig. 22.
which was identified by the double immunocytoche-
mical method using antisera to GH and PRL. GH
was labelled with small gold particles and PRL was
labelled with large gold particles. Bar=500 nm.
rats. The data of the relative proportion of MS
cells shown above could not be directly compared,
because the mode of data description was different
between the two reports.
Chatelain et al. [12] immunocytochemically
observed MS cells in rats at 21 days of fetal age. In
neonatal rats, MS cells were found and these cells
resembled the type III cells of GH or PRL cells
(Fig. 22). The frequency of occurrence of MS cells
during the neonatal period was not so high as
reported by Hoeffler er al. [33]. In fetal mice at
15.5 days of gestation, a few pituitary cells colocal-
ized GH- and PRL-mRNA, but the majority of
cells containing PRL-mRNA did not express GH-
mRNA [16].
3. Developmental and physiological significance
of MS cells
Several possibilities were presented to explain
the significance of MS cells. (i) One is that MS
GH and PRL Cells in the Rat 919
cells are a transitional cell type for the conversion
of GH cells to PRL cells, or PRL cells to GH cells.
(ii) Another possibility is that MS cells are pro-
genitor cells for GH and PRL cells [135]. (iti) The
other possibility is that the MS cell is an independ-
ent type of cell, and may be terminally differenti-
ated. Analysis of the data which had been re-
ported so far, and possible future data may clarify
the genesis and physiological roles of MS cells.
The following reports are favorble to the tran-
sitional cell hypothesis. GH cells appeared earlier
than PRL cells in rats and humans [14, 149]. In
fetal mice, GH synthesis preceded PRL synthesis
[17]. Stratmann and Ezrin [150] previously showed
the possibility of the transition of GH cells to PRL
cells by estrogen treatment using both electron
micriscopy and autoradiographical detection of
3[H]-thymidine uptake. They stated that some of
the previously existing GH cells proliferated and
were converted into PRL cells. Frawley’s group
had reported a large amount of evidence for the
transition from GH cell to PRL cell, or PRL cell to
GH cell using a hemolytic plaque assay [33, 38,
151, 152]. Using transgenic mice, Borrelli et al.
[153] clearly showed that some stem-PRL cells
were derived from part of the stem-GH cells,
stating that PRL cells originated from the GH cell
lineage. One of the transcription factors for GH
and PRL genes was the same, GHF-1 or Pit-1 [6,
154-156]. GH and PRL molecules were consid-
ered to be derived from a common ancestor mole-
cule [157, 158]. Lira et al. [156] suggested that
thyroid stimulating hormone-secreting cells (thy-
rotroph) as well as GH and PRL cells are derived
from a common lineage of pituitary cells.
The analysis of factors of the transition of one
type to the other type is required. Borreli et al.
[153] stated that estrogen is essential for the gene-
sis of stem-PRL cells from stem-GH cells in mice.
Insulin inhibited GH synthesis and secretion [159],
and also reduced the number of fetal GH cells in
vitro [160]. On the contrary, insulin stimulated
PRL synthesis through the activation of a PRL
gene promoter [161]. Inoue et al. [162] recently
induced the transition of GH secreting cells to
PRL secreting cells by insulin or insulin-like
growth factor (IGF-1) in their newly established
pituitary clonal cell line [163]. Thus, insulin, and/
or IGF-1, is closely associated with the develop-
ment of GH and PRL cells, and probably MS cells.
If stimulation of PRL synthesis and secretion can
induce the transition from GH cell to PRL cell,
which may occur through the transitional cell of an
MS cell, excessive stimulation of PRL secretion
may enhance the occurrence of MS cells. Our
preliminary study showed that estrogen treatment
(50 ~g for 3 days) increased the number of MS
cells (Fig. 22) about twice in the neonatal male
rats. Similarly, estradiol increased the proportion
of MS cells in a monolayer culture of male pitui-
tary cells [164].
Chronic stimulation of GHRH using transgenic
mice caused a hyperplasia of MS cells [165],
although some of the MS cells in those transgenic
mice were morphologically similar to those in adult
mice, but others were morphologically different,
and relatively close to those in adenomatous hu-
man pituitaries. Provided that the MS cell is the
common progenitor cell, and this progenitor cell of
MS cells exists even in adult pituitaries, MS cells in
adenomatous tissues may be derived from unreg-
ulated proliferation of preexisting MS cells.
The volume of data accumulated so far seems to
strongly support the theory that the MS cell is a
transitional cell from a GH cell to a PRL cell.
However, it is probable that PRL cells transform
to GH cells through MS cells as shown in Porter et
al. [151, 152]. If such bidirectional conversion
between GH cells and PRL cells occurs in rat
pituitaries, MS cells may be the common progeni-
tor cells. Currently, further study is still needed to
determine which possibility stated above holds for
the rat pituitary.
IV. CONCLUSIONS
A large amount of evidence indicated that the
GH cell and PRL cell populations were morpholo-
gically and functionally heterogeneous. A correla-
tion between morphologically and functionally
different subtypes remains to be studied. Analysis
at a single cell level is needed for further clarifica-
tion. Multiple molecular variants of GH and PRL
have been reported. It is probable that such
molecular variants of the two hormones may play
different physiological roles.
920 S. TAKAHASHI
Heterogeneity of pituitary GH and PRL cells at
different levels (morphological, functional and
molecular) may be the integrated outcome of
various differences in the molecular variants of
hormones, the intracellular age of hormones, the
difference in receptors on the pituitary cells for
hypothalamic regulatory hormones, the difference
in mechanisms of intracellular signal transduction,
and the location of pituitary cells in the gland. The
maturating process of GH and PRL cells may be
associated with these heterogeneities. Heter-
ogeneity of GH and PRL cells in itself alters with
age: the relative proportion of each subtype of GH
and PRL cells changed with age. Age-related
changes in GH and PRL secretion were studied,
and the decrease in GH and PRL syntheses were
clearly explained.
MS cells may be the transitional cell between
GH cells and PRL cells, or a common progenitor
cell of GH and PRL cells. Further study is needed
to clarify the significance of MS cells. From the
Committed
progenitor cell
on 8;
view of the developmental and maturating process
of pituitary cells, the hypothetical schema for
explaining the morphological heterogeneity of GH
and RPL cells, and the relationship among GH,
PRL and MS cells is described (Fig. 23).
The overview of heterogeneities of GH cells and
PRL cells gives the impression that such a wide
spectra of heterogeneities, at various levels from
the molecular to the pituitary level, could probably
be highly helpful for endocrine functions. Such
heterogeneity can give the pituitary gland enough
flexibility to respond to any demands for hormone
secretion.
ACKNOWLEDGMENTS
The author would like to express cordial thanks to Dr.
K. Wakabayashi, Gunma University (Maebashi, Japan)
for kindly supplying of the antisera against GH and PRL,
and to Dr. J. A. Martial, University of Liege (Belgium)
for the rat GH cDNA and rat PRL cDNA. The author is
also grateful to Dr. S. Raiti, the National Hormone and
oF BRU
STATA Sr Obes (Estrogen)
Typell
GH cell Type Il
bre Androgen (Mammosomato troph) Eee
Pluripotent
stem cell . PRL
© Af
Simple duplication\ y pe I type ll
ot Simple duptication Estrogen
ae “ y
\ © LHIFSH &
Simple duplication ——
imple dupli
© (GHRH)
TSH cell
ACTH cell
Fic. 23.
Typel
GH cell
Possible relationship among GH, PRL and MS cells.
(Estrogen)
Simple duplication
(Eatrogen) PRL cell
Pituitary glands consist of GH cells, PRL cells,
gonadotrophs (LH/FSH cells), thyrotrophs (TSH cells), corticotrophs (ACTH cells) and folliculo-stellate cells
(not shown in the figure).
development of GH
the mode of self-duplication.
Pituitary specific transcription factor GHF-1 is known to be involved in the
and PRL cells, and probably TSH cell.
androgen, and that of PRL cells by estrogen. This conversion may be bidirectional.
GHRH stimulates the mitosiss of GH cells, and estrogen that of PRL cell.
Mammosomatotrophs (MS cells) may be the transitional cells between GH cells and PRL cells.
reports, part of PRL cells may be derived from part of GH cells.
The conversion of GH cells is stimulated by
Pituitary cells proliferate by
From several
This transition from stem-GH cells into
stem-PRL cells may probably be stimulated by estrogen.
GH and PRL Cells in the Rat 921
Pituitary Program (University of Maryland School of
Medicine, Baltimore, MD, USA) and the NIDDK, NIH
(Bethesda, MD, USA) for the RIA kit. This study was
supported in part by Grants-in-Aid for Scientific Re-
search from the Ministry of Education, Science and
Culture, Japan, and by the Itoh Science Foundation.
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ZOOLOGICAL SCIENCE 9: 925-939 (1992)
© 1992 Zoological Society of Japan
REVIEW
Chloride Pumps in Biological Membranes
Georce A. GERENCSER! and BLANKA ZELEZNA
Department of Physiology, College of Medicine, University of Florida,
Box 100274, Gainesville, FL 32610-0274, U.S.A.
INTRODUCTION
The electrical activity of isolated biological
epithelia has been a source of intense interest and
much scientific study since the early reports of
DuBois-Reymond [1] and Galeotti [2]. However,
it was not until the brilliant and creative studies of
Ussing [3] on isolated frog skin and, later, those of
Leaf [4] and his co-workers on isolated toad urin-
ary bladder that defined the nature of the bioelec-
tric potential. The defined interrelationship be-
tween bioelectric potential and active Na* trans-
port ushered in the modern era of ion transport
study in epithelia. Skou [5] molecularly defined
the nature of Na* transport with his discovery of
the (Nat +K*)-stimulated ATPase enzyme. For
years thereafter active Na‘ transport across
epithelia has occupied the collective focus of trans-
port physiologists with Cl~ assuming a secondary
role of passive counterion. However, within the
past 20 years there has been an intensive interest in
transmembrane Cl~ transport primarily because
Cl” has been found to move actively in a very wide
range of species [6, 7].
Within the last 20 years three general mecha-
nisms of transepithelial Cl” transport have been
reasonably well established. The first of these is a
strictly passive means of Cl” transport coupled
electrically and/or chemically to primary active
Na®* transport and is exemplified by isolated frog
skin [8] and toad urinary bladder [4]. The second
well-accepted Cl” transport process is secondarily
Received July 3, 1992
' To whom all correspondence should be addressed.
active and is thought to be effected through an
electrically neutral Na*-coupled carrier mecha-
nism which drives Cl” uphill into epithelial cells
via the inward flow of Na* down a favorable
electrochemical potential gradient. This NaCl
symport process is located within the apical mem-
brane if Cl~ is actively absorbed by the epithelium
or is located within the basolateral membrane
(BLM) if Cl~ is actively secreted. Extrusion of
Na* from the cell, therefore maintenance of the
favorable Na* electrochemical potential gradient,
occurs by the ouabain-sensitive (Na*+K‘)-
stimulated ATPase (i.e., primary active Na*
transport) located within the BLM. Epithelia
which exemplify NaCl symport absorption include
prawn intestine [9], flounder intestine [10, 11],
sculpin intestine [12], marine eel intestine [13],
flounder urinary bladder [14], trout urinary blad-
der [15], Necturus gallbladder [16, 17], Necturus
proximal tubule [18], bullfrog small intestine [19,
20], frog skin [21, 22], bovine rumen [23], rat colon
[24], rabbit gallbladder [25], rabbit ileum [26] and
human intestine [27]. Those epithelia in which
Na*-coupled Cl” secretion has been demons-
trated include killifish operculum [28], pinfish gills
[29], shark rectal gland [30], frog stomach [31],
frog cornea [32, 33], rabbit ileum [34] and dog
trachea [35]. In these systems Na * is thought to be
actively recycled at the BLM by the Na* pump
while Cl~ moves energetically downhill from cyto-
sol to the mucosa via a cAMP-enhanced Cl~
conductance [36]. The third widely accepted
epithelial Cl~ transport process is also secondarily
active and involves Cl” /anion antiport and is
found, for example, in anal papillae of mosquito
926 G. A. GERENCSER AND B. ZELEZNA
larvae [37], fish gills [38-40], frog skin [21],
urodele intestine [41], turtle bladder [42], rat intes-
tine [43], rabbit colon [44] and human small intes-
tine [27]. The energy source for this process is
unknown, but it has been suggested that uphill Cl~
transport is energized by a favorable downhill
electrochemical potential gradient for the counter
anion [6].
However, a considerable amount of Cl” trans-
port data has accumulated in the transport litera-
ture that does not conform to any of the three well
established models described above. For instance,
Hanrahan and Phillips [45] have provided evidence
for an electrogenic Cl~ accumulative mechanism
located in the mucosal membrane of locust rectal
epithelium. This mechanism is activated and
stimulated directly by K* and is also independent
of Na* and HCO; . Observations of plant cell
membranes [46, 47], as well as bacterial mem-
branes [48] have yielded Cl -ATPase activity and
associated Cl~ accumulation which are inconsis-
tent with the three models for Cl transport
described previously (vide supra). Perhaps, the
strongest and most compelling evidence for a
primary active transport mechanism of Cl” (Cl~
pump) resides with the observations of Gerencser
[49] and Shiroya et al. [50] who have characterized
Cl~-ATPase activity and ATP-dependent Cl~
transport in the same plasma membrane system as
well as reconstituting these activities in a liposome
system [51]. Indeed, the speculation by Frizzell et
al. [6], Schultz [52], and DePont and Bonting [53]
that Cl~-stimulated ATPases are not involved in
biological Cl~ transport may have been too pre-
sumptuous and premature considering the recent
ground-swell of possible evidence to the contrary.
EXISTENCE AND GENERAL PROPERTIES
Since the time Durbin and Kasbekar [54] first
demonstrated anion-stimulated ATPase activity in
a microsomal fraction of frog gastric mucosa, there
has been little question as to the existence of, at
least, the biochemical manifestation of the en-
zyme. The distribution of anion-stimulated
ATPase activity seems to be as widely distributed
throughout biology as the number of different
plants and animals studied [53, 55, 56].
Anion-stimulated ATPase activity, and there-
fore possibly Cl” pump existence, has been de-
monstrated in both microsomal and mitochondrial
fractions of many tissues (Table 1) in which
HCO; , Cl” or H™ transport occurs, suggesting a
transport function for this enzyme. DeRenzis and
Bornancin [57] demonstrated the existence of
Cl” /HCO3 -stimulated ATPase in goldfish gill
epithelia. It was not until this observation that
HCO; -stimulated ATPase activity was linked
with possible primary active Cl” transport, be-
cause Cl” stimulation of this enzyme had not been
previously demonstrated.
As the name of the enzyme implies, it is directly
stimulated by anions, especially HCO; and Cl.
Bicarbonate stimulation of the enzyme has occu-
pied the predominant focus of attention primarily
because of cellular acid-base implications and also
because of possible simultaneous proton secretion
in gastric mucosal systems [53]. However, HCO3~
can be replaced by several other anions, especially
Cl” and the oxy-anions such as arsenate, arsenite,
borate, selenite, sulfate and sulfite [58-61]. As can
be surmised, however, there are considerable dif-
ferences in effectiveness of the various anions in
different tissues [62]. As an extreme example,
glucaronate stimulates ATPase activity in lizard
gastric mucosa [63] while it inhibits, presumably,
the same enzyme in frog gastric mucosa [64]. As
emphasized by Schuurmans Stekhoven and Bont-
ing [55], this species and tissue variability may very
well be caused by affinity differences of the various
anions for the enzyme.
ATP is the preferred substrate for the anion-
stimulated ATPase, with an optimal Mg?* /ATP
ratio ranging from 0.5 to 2.0 [62, 65, 66]. GTP and
ITP are less preferred substrates than ATP for the
anion-stimulated ATPase, whereas UTP and CTP
are slightly hydrolyzed or not hydrolyzed at all by
the enzyme [58, 65].
The divalent cation Mg** is absolutely required
for maximal anion-stimulated ATPase activity, but
inhibits at high concentrations [64], as are also the
case for the cation-stimulated enzymes: (Na* +
K*)-ATPase and (Ca7+ +Mg**)-ATPase. Mn?*
can substitute for Mg** in the gastric mucosal
enzyme [67], but does so to a lesser extent in the
pancreatic enzyme [65]. Generally Na* or K* has
Chloride Pumps in Biology 927
TABLE |.
Some biological tissues in which Cl -stimulated ATPase activity has been
localized to cellular plasma membranes or microsomal fractions
TISSUE SPECIES REFERENCE
BACTERIA
Cytoplasmic membrane Halobacteria [99]
PLANTS
Cell membrane Algae [110]
Salt gland cell membrane Salt marsh [122]
ANIMALS
Gill Goldfish [57]
Eel [84]
Trout [85, 51]
Fiddler Crab [89]
Blue Crab [90]
Kidney Rat [68]
Rectum Larval Dragonfly [91]
Locust [96]
Intestine Rat [68]
Aplysia [72]
Mantle Oyster [78]
Spinal Motoneurons Rat [108]
Brain Rat [50]
Embryo Sea Urchin [97]
Lens Cow [98]
little or no effect on the activity [64, 65], but K*
was shown to have a stimulatory effect on the
enzyme in rat salivary glands [61]. NH," appears
to inhibit anion-stimulated ATPase activity [67].
LOCATION
Presently, without question, the greatest argu-
ment regarding Cl” - pump activity is its localiza-
tion within the subarchitecture of cells. It seems
that Cl -stimulated ATPase activity resides in
both microsomal and mitochondrial fractions [53]
of cell homogenates. However, DePont and Bont-
ing [53] and Schuurmans Stekhoven and Bonting
[55] have declared that microsomal or plasma
membrane localization of this enzyme is entirely
due to mitochondrial contamination, hence the
dispute. If Cl~-stimulated ATPase activity is
exclusively of mitochondrial origin, it is very dif-
ficult to conceive a mechanism which is the
ATPase that can drive net Cl” transport across
plasma membranes. On the other hand, if the
Cl -stimulated ATPase is located in the plasma
membrane, it would not be difficult to envision
primary active Cl~ transport by this enzyme analo-
gous to the (Na* +K*)-stimulated ATPase and its
role in the net transport of Na* and K* across the
plasma membranes [5].
Without argument, the primary site of Cl7-
stimulated ATPase activity within plant or animal
cells appears to be in the mitochondria; that is, a
property of the mitochondrial Ht-ATPase [68].
928 G. A. GERENCSER AND B. ZELEZNA
Obviously, the key question is: what is the origin
of the Cl -stimulated ATPase activity of non
mitochondrial organelles? Is it as Schuurmans
Stekhoven and Bonting [55] have dogmatically
stated, that all nonmitochondrial organelles which
exhibit Cl” -stimulated ATPase activity have been
contaminated with the mitochondrial-based en-
zyme, or is there a true, separate and distinct
Cl -stimulated ATPase that is localized within the
cellular plasma membranes, and which therefore
can possibly act as the prime mover of net Cl™
transport between the intracellular and extracellu-
lar space?
Van Amelsvoort et al. [62] provided extensive
evidence via differential and density gradient cen-
trifugation techniques on epithelia from trout gill,
rabbit kidney and rabbit stomach that most, if not
all, anion-stimulated ATPase activity is of mito-
chondrial origin. Their speculative conclusions
negated any plasma membrane anion-stimulated
ATPase localization found in other studies [61, 69,
70] on the basis that the results from these studies
possibly artefactual due to improper
homogenization and density gradient centrifuga-
tion techniques. They stated that excessive or
“drastic” homogenization may inactivate the
mitochondrial anion-stimulated ATPase by release
of the endogenous mitochondrial inhibitory pro-
tein [71], therefore this effect would amplify, in a
were
relative sense, mitochondrial contamination
observed in non-mitochondrial organelles.
However, they did not comment why the
mitochondrial inhibitory protein also would not
inactivate the mitochondrial contaminant, anion-
stimulated ATPase found in non-mitochondrial
organelles. Surprisingly, in the same study Van
Amelsvoort et al. [62] observed low cytochrome
oxidase activity in presumably mitochondrial-rich
rabbit kidney and stated that
cytochrome oxidase was either specifically inacti-
vated, or that loss of the mitochondrial inhibitory
protein led to an exaggerated anion-stimulated
ATPase activity in these fractions. They did not
present data nor did they speculate on how these
fractions of
mechanisms were actuated in light of the apparent
contradiction based on the argument that they put
forth for “drastic” homogenization effects. They
also stated that “drastic” homogenization techni-
ques may yield extremely small submitochondrial
particles which may not reach their equilibrium
position in normal empirically determined times of
density gradient centrifugation, which could also
account for erroneous plasma membrane localiza-
tion of anion-stimulated ATPase activity. It had
been the preceding studies that negated any inter-
pretation, other than anion-ATPase being a prop-
erty of mitochondrial H*-ATPase, that stultified
progress in this most complex research area for a
period extending from the mid-1970’s through the
mid-1980’s.
As suggested earlier (vide supra), there are
numerous examples of those tissues that transport
Cl” whose processes of transfer have been mod-
eled mechanistically, but thermodynamically have
not been rigorously defined or tested. Invoking a
cellular active Cl” transport mechanism on
energetic grounds justifies the search for such a
process in the one cellular organelle that regulates
the transfer of material and information (Cl)
between the external world and intracellular con-
tents, the plasma membrane.
The hallmark study demonstrating, unequivo-
cally, the existence of Cl --ATPase activity in a
plasma membrane system free from any possible
mitochondrial contaminant ATPase was that by
Gerencser and Lee [72]. They presented evidence
which indicated that the BLM of Aplysia foregut
absorptive cells contein Cl -ATPase activity.
Their finding that the BLM subcellular membrane
fraction had a high specific activity in (Na* +
K*)-ATPase, but had no perceptible cytochrome c
oxidase activity and a significantly reduced succinic
dehydrogenase activity, supported this conclusion
(Table 2). The observation that there was very
little NADPH-cytochrome c reductase activity in
the membrane fraction (Table 2) suggested that
the BLM in this fraction were also relatively free
from endoplasmic reticulum and Golgi body mem-
brane contamination [73]. The failure of oligomy-
cin to inhibit Cl --ATPase activity in the BLM
fraction was also consistent with the nonmi-
tochondrial origin of the Cl" -ATPase. Supporting
this contention was the corollary finding that oli-
gomycin inhibited mitochondrial Cl -stimulated
ATPase activity. The finding that efrapeptin, a
direct inhibitor of mitochondrial F,;-ATPase activ-
Chloride Pumps in Biology 929
TABLE 2.
from Aplysia
Distribution of marker enzymes and anion-stimulated ATPase during preparation of gut BLM
S-II
Enzyme Homogenate (Mitochondria) Nene
Total protein (mg) 208.59 + 24.95 15.13 + 1.95 5.47 + 1.09
Na*-K*-ATPase 0.85+0.11 0.25 +0.23 BA oar B10)
5’-Nucleotidase 0.41 +0.17 0.37+0.11 0.89 +0.33
Cytochrome c oxidase 0.50+0.12 0.97 £0.23 ND
Succinic dehydrogenase 22.30+4.90 83.40 + 27.60 4.17+2.40
NADPH-cytochrome c reductase 3.73 £0.28 7.44+1.25 1.32+0.18
Mg>*-ATPase 2.69+0.57 5.10+0.84 9.16+1.82
HCO; -ATPase 3.85 +0.83 7.26+0.91 14.12+2.02
Cl -ATPase 2.51+0.59 2.50+0.79 5.77 £2.26
Values are means+SE from 9-11 different preparations.
Enzyme activity is expressed as ~mol-h '-mg
protein.' for Na*-K*-ATPase and 5’-nucleotidase; Alog (ferrocytochrome c)-min '-mg protein! for
cytochrome c oxidase; ymol-min '-mg protein ' for succinic dehydrogenase; nmol-min‘-mg protein! for
NADPH-cytochrome c reductase; “mol-15 min~'-mg protein _' for Mg**-, HCO, , and Cl _-ATPase. P,
pellets from 9,500-g centrifugation; S-III, 40-50% sucrose interface; ND=not detectable.
was ~1.5g. Conditions for enzyme assays were as described in text.
permission.
ity [74], significantly inhibited Mg**-ATPase
activity in the mitochondrial and not in the BLM
fraction [72] unequivocally supported the notion
that the plasma membrane fraction is of extrami-
tochondrial origin. Additionally, Gerencser and
Lee [75] showed that vanadate (an inhibitor of
only “P-type ATPases”) inhibited Cl” -ATPase
activity in the purified BLM fraction. Taken
together, all of these observations strongly support
the hypothesis that Cl -stimulated ATPase activ-
ity exists in, at least, one subcellular locus other
than mitochondria. It appears that in numerous
biological cells, which transport Cl”, Cl -sti-
mulated ATPase activity forms an integral part of
the plasma membrane [68], [76-78], and Table 1.
FUNCTION
To impart a direct role of Cl~ transmembrane
transport to an ATPase, the ATPase should be
shown to be an integral component of the plasma
membrane surrounding the cell periphery. The
energy for active transport of Cl~ can therefore, in
principle, be obtained from the hydrolysis of ATP.
Both of these prerequisites have been amply sa-
tisfied (Existence and General Properties section
Starting gut mucosa
Table from Gerencser and Lee [72] with
and Table 4). Therefore, the next question that
need be asked is: Is the anion-stimulated ATPase
identical with a primary active transport mechan-
ism (“pump”) for anions? Hopefully the following
discussion can lend some insight into this most
controversial question [6, 7, 52, 53, 79].
Countertransport of Cl” and HCO3~ has been
reported in molluscan neurons [80, 81] and mouse
soleus muscle [82] that is sensitve to 4-acetamido-
4’-isothiocyanostilbene-2,2’-disulfonic acid (SITS)
and is not inhibited by thiocyanate in mouse
soleus. It has also been reported in numerous
epithelia [7] that this anion exchange process exists
and is sensitive to the stilbene derivatives. The
stilbene-sensitive countertransport or exchange
mechanism does not seem to require ATP and,
therefore, in all probability, is not an ATPase [83].
It was not until the following observations that
HCO; -stimulated ATPase activity was linked
with Cl” pumping because no Cl activation of
this enzyme had been observed. DeRenzis &
Bornancin [57] were the first to demonstrate the
membrane presence of a (Cl +HCO; )-
stimulated ATPase in goldfish gill epithelium and
suggested that the enzyme could participate in the
branchial Cl~/HCO3° exchange mechanism.
930, G. A. GERENCSER AND B. ZELEZNA
Bornancin et al. [84] confirmed these results in
freshwater eel gill epithelium as did Bornancin et
al. [85] in freshwater trout gill epithelium. Kinetic
studies in these three gill epithelial systems strong-
ly suggested that a (Cl /HCO3_)-stimulated
ATPase is involved in the Cl” /HCO3” exchange
mechanism and therefore in the acid-base regula-
tion of freshwater fish. These authors reported a
parallelism between the affinities of the ATPase
for Cl” and both the Cl” affinity for the gill
transport mechanism and the Cl” influx rate. The
affinity constants for the Cl” -stimulated ATPase
were 1.0, 5.9 and 23.0 meq/L for the goldfish [57],
freshwater trout [85], and freshwater eel [84] gill
epithelium, respectively. The affinity of Cl” for
the transport systems in vivo was 0.07, 0.25 and 1.3
meq/L for the goldfish [57], freshwater trout [85]
and freshwater eel [84] gill epithelium, respective-
ly, while the corresponding maximal Cl~ influxes
were 55.0, 19.6, and 0.36 weq/hr/100 g. In addi-
tion, the finding that Cl” activation of anion-
stimulated ATPase activity was inhibited by
thiocyanate [57] was consistent with transport stu-
dies which showed that Cl~ influxes were inhibited
by thiocyanate [86]. These studies on gill epithe-
lium strongly support the hypothesis that the Cl” -
stimulated ATPase is involved in gill anion ex-
changes that are related to mineral and acid-base
homeostasis in freshwater fish.
The fiddler crab gill has been shown to actively
absorb Cl” from low salinities [87] and actively
extrude Cl” in high-salinity media [88]. In concert
with these findings DePew & Towle [89] demon-
strated the existence of an anion-stimulated
ATPase in the gill cell plasma membrane of fiddler
crab and suggested that this enzyme is so situated
with its environment that it is highly accessible to
Cl” and HCO; _, and thus many play a direct role
in acitve Cl” /HCO3 exchange.
Lee [90] used an additional approach to the
question concerning correspondence between
transport and anion-stimulated ATPase activity.
After it was established that anion-stimulated
ATPase activity existed in the plasma membrane
of blue crab gill epithelium, the animals were
adapted to low salinities. This thinking presumed
that Cl” /HCO3” exchange should increase under
these osmotic stressful conditions, therefore this
transport activity should be reflected in an increase
in the activity of anion-stimulated ATPase activity.
This was indeed the case and Lee [90] suggested
that anion-stimulated ATPase activity appears
likely to play an important role in anion transport
for osmoregulatory and/or acid-base homeostasis
in marine organisms.
Komnick et al. [91] reported the presence of
(Cl” +HCO3 )-stimulated ATPase activity in
plasma membranes of larval dragonfly rectum.
The Cl -stimulated ATPase activity was inhibited
by thiocyanate as was the Cl” influx into the rectal
epithelia. These results suggested the possible
existence of an ATPase-mediated, active Cl~
transport mechanism located in the plasma mem-
brane of larval dragonfly rectal epithelial cells.
In the eel (Anguilla japonica) intestine, elec-
trophysiological experiments have shown that ac-
tive transport of Cl” coupled with water transport
markedly increases during seawater adaptation
[92, 93]. The observed increase in Cl” absorption
raised the question of an associated increase in
activity of an enzyme contributing to the transport
process. It was demonstrated by Morisawa and
Utida [94] that anion-stimulated ATPase activity
existed in an oligomycin-insensitive, thiocyanate-
sensitive membrane fraction of eel intestinal en-
terocytes that was also relatively deficient of
cytochrome oxidase activity. Seawater adaptation
increased the enzyme activity commensurate with
changes in Cl” and water transport. From these
considerations, these authors concluded that the
anion-stimulated ATPase played a direct role in
Cl” transport in the eel intestine.
The hindgut of the desert locust possesses an
unusual chloride transport system [95]. The iso-
lated locust rectum absorbs chloride from the
mucosal (lumen) to the serosal (haemolymph) side
at a rate which is equal to the short-circuit current
(I,.). Net chloride transport (J$%,) persists in
nominally Na-free or HCO 3(CO3)-free saline, is
insensitive to normal inhibitors of NaCl co-
transport and anion exchange, and is independent
of the net electrochemical gradient for sodium
across the apical membrane. However, active
chloride transport is strongly dependent on mucos-
al potassium (K,=5.3mM-K). Chloride entry
across the apical membrane is active, whereas the
Chloride Pumps in Biology 931
net electrochemical gradient across the basal mem-
brane favors passive Cl exit from the cell.
Although mucosal potassium directly stimulates
“uphill” chloride entry, there is no evidence for
coupled KCl co-transport, nor would co-entry with
potassium be advantageous energetically.
This Cl~ absorption is electrogenic, not depen-
dent on Na* or HCO; /COs, and insensitive to
inhibitors of NaCl cotransport or HCO; /Cl~
exchange [96]. To determine if active Cl” trans-
port across rectal epithelia might be due to an
anion-stimulated ATPase, a microsomal fraction
was obtained by differential centrifugation. Micro-
somal ATPase activity was stimulated in the fol-
lowing sequence: sulphite >bicarbonate >chlor--
ide. Maximal ATPase activity was obtained at 25
mM HCO3° or25mM Cl. Thiocyanate (10 mM)
inhibited 90% of the anion-stimulated ATPase
activity. The microsomal fraction was enriched in
the plasma membrane markers, leucine ami-
nopeptidase, alkaline phosphatase, 5’-nucleo
tidase, and gamma-glutamyltranspeptidase, and
had little contamination of the mitochondrial en-
zymes, succinate cytochrome c reductase and
cytochrome oxidase. Na, K-ATPase was enriched
in the mitochondrial fraction. Microscopic ex-
amination confirmed that basolateral membranes
were associated with mitochondria following dif-
ferential centrifugation, while the microsomal frac-
tion contained little mitochondrial contamination.
These results indicate the presence of an anion-
stimulated ATPase activity that could be responsi-
ble for active Cl~ transport across locust recta.
In cultured cells derived from isolated micro-
meres of sea urchin eggs, Cl’ /HCO3 -ATPase
activity was found in the plasma membrane and
the microsome fractions before and after the initia-
tion of spicule formation [97]. After initiation, the
skeletal vacuole fraction was obtained from sub-
cellular structures containing specules. Cl 7/
HCO; -ATPase in the skeletal vacuole membrane
probably mediates HCO; transport into the
vacuoles to supply HCO; for spicule formation.
An anion-stimulated ouabain-insensitive Mg? *-
ATPase activity has been found in fresh homogen-
ates prepared from capsules and epithelia of
bovine lenses [98]. Approximately equal activity
was observed in the presence of HCO; or of Cl.
The stimulation of each anion obeys saturation
kinetics, with an optimum at approximately 20
mM Cl” or HCO; _. In keeping with other tissues,
the diuretic drugs furosemide and ethacrynic acid
are inhibitory. ATP is the primary substrate for
the enzyme, which also shows some activity on
GTP, ITP, and even ADP. Little Na‘/K*-
dependent ATPase activity was observed in the
fresh homogenate, but it increased in lyophilized
preparations. In contrast, the lyophilized prepara-
tions showed no anion-dependent ATPase activity.
It is postulated that active bicarbonate ion trans-
port in the lens may be mediated by this anion-
dependent ATPase.
Halorhodopsin [99, 100], one of the retinal
proteins in the cytoplasmic membrane of halobac-
teria, is an inward-directed light-driven elec-
trogenic pump for chloride ions that generates an
inside-negative membrane potential similar to that
of bacteriorhodopsin, which transports protons out
of the cell interior. However, the physiological
role of halorhodopsin might be not only to gener-
ate a transient proton-motive force on illumina-
tion, but also to maintain cell volume [101]. This is
because in these organisms the high (several mo-
lar) external NaCl concentration in the medium is
balanced mostly by intracellular KCI, and although
the replacement of Na* with K* can be accom-
plished, as in many other systems, by a combina-
tion of a K*/Na° antiporter [102, 103] and elec-
trogenic K* uptake [104, 105], the net uptake of
Cl” requires an active accumulation system. In-
deed, as with protons in the case of bacterio-
rhodopsin, a second transport pathway for active
Cl transport exists in the dark [106], apparently
driven independently, by proton-motive force.
The elements of the foregoing hypothesis can be
observed in vesicles prepared from Halobacterium
halobium cell envelopes containing halorhodopsin
[107]. Thus, in the absence of K* (e.g., in 3M
NaCl), illumination causes the inward flow of Cl",
which is detectable by direct determination of the
accumulated Cl in vesicles equilibrated first with
Na SO, or phosphate. When the illumination is
started, there is an initial passive influx of protons,
which slows as a concentration gradient for pro-
tons (inside acid) develops. During this time, Na*
takes over as the main counterion to the Cl”
932 G. A. GERENCSER AND B. ZELEZNA
movement. Once the protonmotive force
approaches zero, the net proton flux ceases, and
light will drive the continued uptake of NaCl
instead. Indeed, illumination is seen to cause
swelling of the vesicles, particularly when gramici-
din is added to increase the electrical potential-
driven secondary Na‘ uptake.
Halorhodopsin, as bacteriorhodopsin, requires
no other component than the opsin, a small (MW
above 26,000) integral membrane protein, and the
retinal, for the light-driven transport. In both
proteins the retinal is attached to a lysine via a
protonated Schiff base. The intimate association
of the retinal with various amino acid residues in
halo-opsin is indicated by the fact that the
wavelength maximum of the pigment is shifted
from 440 nm, that of a protonated retinal Schiff
base in solution, to 578 nm. Thus, halorhodopsin,
as bacteriorhodopsin, is a purple protein. Absorp-
tion of a photon causes the isomerization of the
retinal from all-trans to 13-cis; this initiates a
sequence of thermally driven reactions which lead
back to the parent pigment in a few tens of
milliseconds (the “photocycle”). This character-
ization provides the first strong evidence for the
existence of a Cl pump mechanism residing in
bacteria.
The following studies on rat brain motoneurons
provided the strongest evidence in vertebrates for
the existence of a Cl” pump. Shiroya eft al. [50]
demonstrated that EDTA-treated microsomes
prepared from rat brain mainly consisted of sealed
membrane vesicles 200-500 nm in diameter and
were rich in both Cl--ATPase and Na”, K’*-
ATPase activities. Such Cl” -ATPase-rich mem-
brane vesicles accumulated Cl~ in an ATP-
dependent and osmotically reactive manner in the
presence of 1 mM ouabain. The Cl” uptake was
maximally stimulated by ATP with a K,, value of
1.5 mM; GTP, ITP, and UTP partially stimulated
Cl” uptake, but CTP, beta, gamma-methylene
ATP, ADP, and AMP did not. The ATP-
dependent Cl” uptake was accelerated by an in-
crease in the medium Cl~ concentration with a K,,
value of 7.4mM. Such stimulation of Cl” uptake
by ATP was dependent on the pH of the medium,
with an optimal pH of 7.4, and also on the
temperature of the medium, with an optimal range
of 37-42°C. Ethacrynic acid dose-dependently
inhibited the ATP-dependent Cl” uptake with a
concentration for half-maximal inhibition at 57
uM. N-ethylmaleimide (0.1 mM) completely inhi-
bited and sodium vanadate (1 mM) partially inhi-
bited the ATP-dependent Cl” uptake. The mem-
brane vesicles did not accumulate H~ in the Cl—
uptake assay medium. The ATP-dependent Cl—
uptake profile agreed with that of Cl--ATPase
activity reported previously [108], and this strongly
supports the idea that Cl” -ATPase in the brain
actively transports Cl.
Gradmann and his colleagues [109] have pro-
vided electrophysiological data and ATP synthesis
by the Cl” pump through reversal of Cl~ elec-
trochemical gradients in Acetabularia which pro-
vided strong evidence for the existence of a Cl~
pump in algae. Buttressing these conclusions were
those of Ikeda and Oesterhelt [110], who showed a
Mg?*-ATPase, isolated from Acetabularia, recon-
stituted into liposomes and tested for a Cl -
translocating activity. A significant increase in
°°Cl~ efflux from the negative and neutral lipo-
somes was observed by addition of ATP in the
presence of valinomycin after incorporation of the
enzyme by short-term dialysis. The ATP-driven
°°CI~ efflux was inhibited by addition of azide, an
inhibitor of the ATPase. When chloride was
replaced by sulfate, no ATP-dependent sulfate
efflux was detectable from the proteoliposomes.
Proton-translocating activity of the enzyme was
also tested, and no fluorescent quenching of 9-
ACMA was observed. These observations strong-
ly suggested the existence of a Cl” pump in
Acetabularia.
Graves and Gutknecht [111] have provided evi-
dence for an electrogenic Cl” pump with similar
properties in the membrane of Halicystis, another
marine alga that is related to Acetabularia. For the
question of the physiological significance of the
electrogenic Cl” pump in Acetabularia, Gradmann
[109] favors a “Mitchellian” answer. This primary,
electrogenic ion pump would create an electroche-
mical driving force to fuel secondary, elec-
trophoretic (or electroneutral) transport proces-
ses, such as uptake of sugars or amino acids.
However, the most rigorous and definitive proof
for a Cl” pump’s existence and its mode of opera-
Chloride Pumps in Biology 933
tion rests with the following group of experiments
by Gerencser and his colleagues [56, 72, 109].
Gerencser and Lee [68, 72] presented evidence
which indicated that the BLM of Aplysia foregut
absorptive cells contains Cl” -ATPase activity.
Biochemical properties of the Aplysia foregut
absorptive cells BLM-localized Cl -stimulated
ATPase include the following: 1) pH optimum=
7.8; 2) ATP being the most effective nucleotide
hydrolyzed; 3) also stimulated by HCO;—, SO3”,
and S03”, but inhibited by NO; , and no effect
elicited by NO3” and SO,~7, 4) apparent K,, for
Cl” =10.3 mM while the apparent K,, for ATP=
2.6 mM; and 5) a requirement for Mg** which has
an optimal concentration of 3mM [72]. Coin-
cidentally, Cl~ has an intracellular activity approx-
imating its apparent K,, for the Mg**-dependent
ATPase (vide supra), which supports the notion
for its physiological and biochemical activities.
Additionally, the ATPase activity stimulated by
Cl~ was strongly inhibited by thiocyanate, vana-
date, and acetazolamide but not inhibited by oua-
bain (Table3). These results with inhibitors
strongly suggested a possible participation by the
Cl -stimulated ATPase in net chloride absorption
by the Aplysia gut [72]. The finding that anion-
stimulated ATPase is inhibited by thiocyanate, but
not by ouabain has also been demonstrated in
many tissues known to perform active anion trans-
port and to contain anion-stimulated ATPase
activity [112]. This coincidental inverse parallel
between ouabain insensitivity and thiocyanate sen-
sitivity to Cl” -stimulated ATPase activity and net
TABLE 3.
active Cl” absorption in the Aplysia gut warranted
conjecture that the active Cl” absorptive mechan-
ism could be driven by a Cl -stimulated ATPase
found in the BLM of the foregut absorptive cell.
Additional support for this contention rested with
the finding that Cl” -stimulated ATPase activity of
the BLM is inhibited by vanadate (Table 3).
Vanadate has been shown to inhibit ATPases,
which form high-energy phosphorylated inter-
mediates while having no effect on the mito-
chondrial anion-sensitive ATPase [113]. These
results strongly suggested that the Cl -stimulated
ATPase is an ion-transporting ATPase of the “P”
variety rather than the “Fo—F,” or “V” types.
Acetazolamide inhibited Cl -stimulated
ATPase activity in the Aplysia gut (Table 3). This
finding has also been demonstrated in blue crab gill
HCO; -ATPase [90]. Although acetazolamide
has been shown to be a specific inhibitor of carbo-
nic anhydrase [114], it has also been demonstrated
to be a Cl” transport inhibitor [115]. Additionally,
it has been shown by Gerencser [116] that carbonic
anhydrase does not exist in the BLM of the Aplysia
gut absorptive cell. Thus, the data further
strengthen the notion that the Cl -stimulated
ATPase, which is inhibited by acetazolamide, may
be involved in net Cl~ transport across the mollus-
can gut.
Furthermore, Gerencser and Lee [75] demon-
strated an ATP-dependent Cl” uptake in Aplysia
inside-out gut absorptive cell BLM vesicles that
was inhibitable by thiocyanate, vanadate, and also
by acetazolamide. The ATP-driven Cl” uptake
Effects of Inhibitors on Cl -ATPase Activity
alsibition Concentration
Specific Activity %
(Gay Me?* +Cl--ATPase Cl--ATPase LQMDLOR
Control 16.8+0.3 6.7 0
Thiocyanate 10.1 7.7+0.6 4.5 33
Acetazolamide 1.0 11.1+1.0 1.4 79
Acetazolamide 2.0 VW se 20) 0.4 94
Ouabain 1.0 16.5+0.5 7.0 0
Vanadate 0.5 14.0+1.4 4.4 34
Vanadate 1.0 10.5+1.1 2.5 63
Values are means+SE from 3-5 different experiments.
Table from Gerencser and Lee [72] with permission.
protein” '. Inhibitors were as described in text.
Specific activity is expressed as ~mol-15 min” '-mg
934 G. A. GERENCSER AND B. ZELEZNA
TABLE 4. Effect of ATP on Transport Parameters in Basolateral Membrane Vesicles
Experimental Condition
Cl~ Uptake
(nmol/mg protein)
Vesicular Membrane
Potential
Difference (mV)
+ATP 102.7+7.9
—ATP A) 7] 3ES)9)
+Nonhydrolyzable ATP analog 59.6+8.3
(5’-adenylyl-imidodiphosphate)
NO; for Cl” (mole for mole)
Values are means + SE; n=number of experiments.
was obtained in the absence of Na’, K*, HCO; ,
or a pH gradient between the intra- and ex-
travesicular space, which is strong suggestive evi-
dence that the Na*-K*-ATPase enzyme, Na™ /
Cl~ symport, K*/Cl~ symport, Na*/K*/Cl7
symport, Cl” /HCO3 ° or Cl /OH antiport and
K*t/H* antiport were not mechanisms that are
involved in the accumulation of Cl” within the
vesicles.
To further elucidate the electrogenic nature of
the ATP-dependent Cl~ transport porcess, several
experimental maneuvers were performed by
Gerencser [49] as follows. First, an inwardly
directed valinomycin-induced K* diffusion poten-
tial, making the BLM inside-out vesicle interior
electrically positive, enhanced ATP-driven Cl~
uptake compared with vesicles lacking the
ionophore. Second, an inwardly directed FCCP-
induced H* electrodiffusion potential, making the
BLM inside-out vesicle interior less negative, in-
creased ATP-dependent Cl~ uptake compared to
control. Third, ATP increased intravesicular nega-
tivity measured by lipophilic TPMP™~ distribution
across the vesicular membrane (Table 4). Addi-
tionally, both ATP and Cl” appeared to be neces-
sary for generating the negative intravesicular
membrane potential, because substituting a
nonhydrolyzable ATP analog for ATP, in the
presence of Cl” in the extravesicular medium, did
not generate a potential above that of control
({56], and Table 4). Likewise, substituting NO3~
for Cl~ in the extra- and intravesicular media, in
the presence of extravesicular ATP, caused no
change in potential difference above that of con-
trol (Table 4). These results also suggested that
hydrolysis of ATP is necessary for the accumula-
3 = 34! Q2e 2.5 12
3 0.0+5.2 12
3 = 1.3)25(0).) 12
+3.0+4.6 3
Table is taken from Gerencser, et al. [109] with permission.
tion of Cl” in the vesicles. Furthermore, vana-
date, acetazolamide, and thiocyanate inhibited the
(ATP+Cl~)-dependent intravesicular negativity
[109]; and in addition, it had been demonstrated
that the pH optimum of the Cl -stimulated
ATPase [72] coincided exactly with the pH opti-
mum of 7.8 of the ATP-dependent Cl” transport
in the same fraction of Aplysia foregut absorptive
cell BLM vesicles [49]. Therefore, both aspects of
the Cl” pump (ATPase and ATP-dependent Cl—
transport) have the same pH optimum, which
suggests that these properties are part of the same
molecular mechanism.
SULFHYDRYL LIGANDS OF Cl” PUMP
It appears that the catalytic, Cl -stimulated
ATPase activity, and its corollary transport com-
ponents, ATP-dependent Cl~ transport and ATP-
detendent Ay in the BLM of Aplysia foregut
absorptive cells are dependent on intact sulfhydryl
ligands [117, 118]. P-chloromercuribenzensul-
fonate (PCMBS) forms a mercaptide complex with
sulfhydryl ligands of the Cl” pump which inhibit
Cl" -stimulated ATPase activity [117], ATP-
dependent Cl” accumulation and ATP-dependent
4y in BLM vesicles [118]. These catalytic and
transport inhibitions of Cl” pump activity are
totally reversed by dithiothreitol (DTT), which is a
specific thiol reducing agent [119]. This result
provides strong evidence that the ligands involved
in both hydrolysis of ATP and accumulative Cl—
transport are sulfhydryl and not carboxyl, phos-
phoryl, tyrosyl or amino [119]. In addition, it
appears that the sulfhydryl ligands of the Cl ~
pump that are responsible for its catalytic and
Chloride Pumps in Biology 935
transport activities are located on the cytoplasmic
surface of the BLM of Aplysia gut absorptive cells,
for PCMBS has been shown to have a very low
lipid solubility [119], and this restricts its action to
surface and not intramembranous sulfhydryl
ligands.
RECONSTITUTION OF THE Cl” PUMP
Reconstitution of a membrane protein into a
liposome provides one of the few methods needed
to rigorously demonstrate the existence of a sepa-
rate and distinct biochemical and physiological
molecular entity. This method also provides evi-
dence that all components of the solubilized pro-
tein haye been extracted intact. With this premise
in mind, Gerencser [51] reconstituted both aspects
of the Cl~ pump; that is, the catalytic (ATPase)
and transport components from the BLM of Aply-
TABLE 5.
(A) Proteoliposome ATPase Activity
sia gut absorptive cells, as shown in Table 5. Table
5 shows Cl -stimulated ATPase activity exists
significantly (P<0.05) above Mg’*-stimulated
ATPase activity found in the proteoliposome
population extracted and generated with digitonin.
Vanadate (0.1 mM) inhibited this Cl -stimulated
ATPase activity by 99%. From this digitonin-
generated proteoliposome population, it is also
seen in Table 5 that there is a significant ATP-
dependent Cl~ uptake into these proteoliposomes
above that of control (P<0.05), and that this
ATP-dependent Cl~ uptake is also inhibited by 0.1
mM vanadate. Not detected in the proteolipo-
somes solubilized and formed by digitonin were
Na‘ /K+t-ATPase, alkaline phosphatase, (Ca** +
Mg?*)-ATPase, or cytochrome c oxidase activities
and, coupled with a previous observation that
FCCP had a stimulatory and not an inhibitory
effect on ATP-driven Cl” accumulation in the
Reconstitution of Cl” Catalytic and Transport Activities
Extractive and Mg?t- (Moat Glp)s wanl(Mezn Gln)
reconsitutive ATPase ATPase ATPase
detergents + vanadate
Cholate n.d. n.d. —
Octyl glucoside n.d. n.d. —
Lubrol PX n.d. n.d. _
Digitonin 2.8+0.4 11.2+2.0 2.9+0.4
(B) Cl Uptake Into Proteoliposomes
Extractive and —ATP + ATP +ATP
reconstitutive + vanadate
detergents
Cholate 87.5+5.6 82.7+6.9 80.6+8.3
Octyl glucoside 82.7+8.0 73.6+9.2 SO) SE(}.0)
Lubrol PX 28.3+11.1 39.Q3E 13},9) 39.3+ 14.0
Digitonin 91.2+6.0 192.5+9.3 93.1+7.9
Values are means+SE from four individual determinations.
Conditions for the enzyme assay are described in Materials
Time period of assay for V; determined previously. Vanadate (0.1 mM) had no significant effect
Either vanadate (0.1mM) was preincubated with the proteoliposomes in the
mg protein for Mg**- and (Mg** +Cl_)-ATPase.
and Methods.
on Mg**-ATPase activity.
Enzyme activity is expressed as ~mol/15 min per
reaction mixture (50 1 containing 10 mM imidazole-HCl, 250 mM sucrose, 3 mM MgSO,, and 25 mM choline
chloride) at pH 7.8 for 10 min at 25°C, or 5mM ATP was added to the reaction mixture to initiate the incubation
for the transport experiments.
The incubation for the uptake of *°Cl~ was measured for 10 min at 25°C. Time
for steady-state values for both ATP-independent and ATP-dependent *°Cl~ uptakes was based on previous
observations. *°Cl~ uptake is expressed as nmol/mg protein.
reaction mixture; — represents its absence; n.d., not detectable.
permission.
+ Represents a compound’s presence in the
Table is taken from Gerencser [51] with
936 G. A. GERENCSER AND B. ZELEZNA
BLM vesicles, it is suggested that none of these
enzymes nor eukaryotic vacuolar H‘-ATPases
could express Cl” pump activity. These data also
suggested that these two major observations are
manifestations of one molecular mechanism: the
Cl~ pump. Support of this contention rested with
the findings that vanadate (an inhibitor of P-type
ATPases) inhibited both Cl” -stimulated ATPase
activity and ATP-dependent Cl” transport in the
digitonin-based proteoliposomes (Table 5). Even
though Krogh [120] first coined the term “Cl—
Pump,” it was not until the reconstitution of all of
its components into an artificial liposomal system
through the study mentioned above [51] that the
existence of this mechanism (primary active trans-
port mechanism) was rigorously proven.
MOLECULAR WEIGHT OF THE Cl" PUMP
Utilizing sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) techniques to
digitonin-generated proteoliposomes containing
the Cl” pump components from Aplysia gut
absorptive cells as shown previously [51], the
approximate molecular weight of the Cl” pump
was ascertained. Since both aspects of the Cl~
pump were inhibited by vanadate (Table 5), it was
surmised that the approximate molecular weight of
the Cl” pump of Aplysia gut BLM should be
approximately 100 K daltons since vanadate only
inhibited “P” type ATPases and not “FoF,” or “V”
type ATPases [121]. The alpha-subunit of all “P”
type ATPases approximates 100K daltons in
molecular weight [121]. Two protein bands were
eluted through SDS-PAGE, one of which was a
major band at 116 K daltons and the other being a
minor band at 97.5 K daltons. These two protein
bands were obtained whether 5-mercaptoethanol
was present or not in the buffer medium. This
finding indicates that the Cl” pump of Aplysia is
not dependent upon the integrity of subunit-
linking sulfhydryl ligands. Also, these purified
proteins of the Cl” pump have been subjected to
phosphorylation within the proteoliposome and
the reaction sequence and kinetics of the reaction
sequence of the enzyme have been determined:
Mg?* causing phosphorylation, Cl~ causing de-
phosphorylation, and all in a time frame consistent
with an acyl phosphate linkage. Hydroxylamine
and high pH destablize this phosphorylation.
Orthovanadate (10~’M) almost completely in-
hibit the Mg**-driven phosphorylation reaction.
CONCLUSIONS
In summary, it is quite apparent that in the past
few years there has been an increasing number of
convincing studies in a variety of animal tissues
that have provided indirect, correlative evidence
that active Cl” transport is primary by nature. The
active translocation of Cl” by an enzyme that
directly utilizes the energy from ATP hydrolysis is
not unlike that observed in plants [122, 123].
Indeed the evidence for primary active Cl” trans-
port in these simple living things is almost as
convincing as that presented for (Na* +K7~)-
stimulated ATPase and (Ca** +Mg?*)-stimulated
ATPase in their respective roles for actively trans-
ferring Na*, K*, and Ca?* across animal plasma
membranes. As alluded to by DePont and Bonting
[53], future experimental steps in assuring that an
animal Cl -stimulated ATPase is involved in pri-
mary Cl transmembrane movement should
approximate the following: 1) a specific inhibitor
for the enzyme should be found or synthesized
(e.g., an antibody), and this inhibtor should be
shown to inhibit the transport process; and/or 2)
the Cl -stimulated ATPase should be biochemi-
cally isolated or purified and after its incorporation
in liposomes should then be shown to support
active Cl” transport. This demonstration of re-
constitution has been shown in the present review
which provided the first direct evidece for the
existence of a new “P” type ATPase: the Cl~
pump. .
ACKNOWLEDGMENTS
The authors wish to acknowledge their gratitude to
their technologists, students, and collaborators for their
able contributions to the studies reviewed and performed
herein. These studies were supported by NIAMD Train-
ing Grant (1-TO1-AM-05697-02 and -03), NIH Grants
(No. AM17361 and RCDA No. AM00367), D.S.R. Seed
Award (NO. 229K15), Whitehall Foundation Grant (No.
78-156 ck-1), D.S.R. Award (No. 122101010), and The
Eppley Foundation for Research, Inc.
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Chloride Pumps in Biology
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ZOOLOGICAL SCIENCE 9: 941-946 (1992)
© 1992 Zoological Society of Japan
HPLC Analysis of Retinoids Extracted from the
Planarian, Dugesia japonica
Katsu Azuma!, Naouiko Iwasakt!, MASAMI AZUMA2,
TAKAO SHINOZAWA°® and Tatsuo Suzuki‘
'Department of Biology, Osaka Medical College, Takatsuki, Osaka 569,
"Department of Health Science, Osaka Kyoiku University, Osaka 547,
3Department of Biologyical and Chemical Engineering, Faculty of
Engineering, University of Gunma, Kiryu, Gunma 376, and
“Department of Pharmacology., Hyogo College of
Medicine, Nishinomiya, Hyogo 663, Japan
ABSTRACT—Retinoids extracted from the planarian, Dugesia japonica were analyzed by high-
pressure liquid chromatography (HPLC). All-trans retinal, all-trans retinol, and all-trans retinyl ester
were detected in the extracts from the head and tail pieces of the worm, while 11-cis retinal was detected
in the extracts from the head pieces. The amounts of all-trans retinal, 11-cis retinal and all-trans retinol
including the retinyl ester were 0.1-1.1, 0.11-0.19, and 20-50 pmol/head, respectively. The planarian
contained many oil-droplets which emitted the green-yellow fluorescence probably derived from retinol
and retinyl ester. These results suggest that the planarian contains all-trans retinol and the retinyl ester
in oil-droplets and 11-cis retinal as the chromophore of the visual pigment in the eye.
INTRODUCTION
The planarian is one of the lowest metazoans.
The eye of the planarian consists of pigmented
cells and photoreceptors of a microvillar type [1].
Extracellular microelectrode recordings from the
eye of the planarian Dugesia tigrina suggested the
presence of a rhodopsin-like photopigment whose
absorption maximum was at about 508 nm [2].
Spectral phototaxis experiments showed the sensi-
tivity maximum of the planarian eye (Planaria
lugubris) at about 475nm [3] and 530nm for
Dendrocoelum lacteum [3]. The differences in
those maxima may have been caused by a con-
tribution from the dermal photoreceptors [2], an
effect of screening pigments [2] or, perhaps, simply
a species difference. At present, the visual pig-
ment of the planarian has hardly been investigated
by spectrophotometric methods, because the
worm contains so little visual pigment. In histo-
Accepted June 8, 1992
Received April 6, 1992
chemical experiments, it has been reported that
the photopigment of the planaria (Dugesia japoni-
ca) is a chromoprotein which possesses retinal-
dehyde as the chromophore [4]. Recently an
immunochemical study suggested the presence of
rhodopsin-like protein in the head of the planarian
Dugesia japonica by use of anti-frog-rhodopsin
rabbit IgG [5].
Although 11-cis-retinal is the most ubiquitous as
the chromophore in the vertebrate and inverte-
brate rhodopsin [6], a variation in the chromo-
phore of visual pigment is found in other spe-
cies: 11-cis 3-dehydroretinal is found in many fresh
water vertebrates [7, 8] and invertebrates [9, 10];
11-cis 3-hydroxyretinal is found in the insects [11];
and 11-cis 4-hydroxyretinal is in a bioluminescent
squid [9]. In addition all-trans retinal and 13-cis
retinal are seen in Halobacterium halobium [12]. It
is unknown whether or not the chromophore of the
visual pigment of the planarian is 11-cis retinal.
The purpose of this study is to estimate the con-
figuration of the chromophore of the planarian
visual pigment by high-pressure liquid chroma-
942 K. Azuma, N. IwasAkI et al.
tography (HPLC) analysis of retinoids extracted
from the worms. Our results indicate that 11-cis
retinal is one of the most plausible candidates for
the chromophore.
MATERIALS AND METHODS
Materials
The planarian worms, Dugesia japonica were
collected from streams in the suburbs of Kyoto city
(Kyoto prefecture, Japan) and Kiryu city (Gunma
prefecture, Japan). Kyoto worms were maintained
by feeding on fresh beef livers, and used for
extractions of retinoids from their whole bodies or
both the head pieces (anterior part containing the
eyes) and the tail pieces (the tissues without the
head). Head pieces of kiryu worms were stored as
frozen materials and used for extraction of re-
tinoids.
Extractions of retinoids
Usually, retinoids were extracted from the fresh
or frozen head pieces by the oxime method which
was developed to extract the retinal from biologic-
al materials as retinaloximes (syn- and anti-forms)
in the original isomeric configuration without ther-
mal isomerization [13, 14]. The planarian samples
were homogenized in a solution of 100mM
NH,OH (pH 7.2) and methanol (final concentra-
tion of methanol was 60-70%) using a homogeniz-
er (Physcotron NS-50, Nichion Irikakikai Seisa-
kusho Co. Ltd., Japan). The homogenate was
mixed with dichloromethane and n-hexane (1:2,
vol/vol), shaken vigorously and centrifuged at
2,500r.p.m. for 15min. The upper layer
(dichloromethane/hexane layer) was collected.
This extraction was repeated three times. The
collected solution was stored as extracts of re-
tinoids.
In a few cases, the planarian samples were
freeze-dried for hexane extraction of retinoids.
The freeze-dried samples were shaken vigorously
in hexane solvent and centrifuged at 2,500 r.p.m
for 15min. The supernatant was collected and
hexane extraction was repeated 3 times. The
collected solution contained retinol and retinyl
ester. Precipitates were gently aspirated to evapo-
rate hexane solvent and served for the extraction
of retinoids by the oxime method as described
above. The obtained solution (dichloromethane /
hexane extract) contained retinaloxime and retinyl
ester. After evaporating solvents of the extracts
mentioned above, the residues were dissolved in
50 ul of hexane/diethylether/ethanol (90/10/0.1,
vol/vol) and analyzed by HPLC. All procedures
were carried out under dim red light.
Detection of retinoids in the samples
Extracts of retinoids from the planarian samples
were analyzed by the HPLC method as reported
previously [15]. An HPLC system equipped with a
4.6250 mm column of YMC-Pack A-003-3 SIL
(Yamamura Chemical Labo. Co. Ltd., Japan) and
a pump (TRI ROTER, JASCO, Japan) was used.
The eluent was a mixture of n-hexane, diethylether
and ethanol (90:10:0.1, vol/vol) and was used at
the flow rate of 1.3 ml/min for 50min. The
absorbances of the fractions at 350 nm and at 280-
500 nm were measured with a detector UVDEC-
100-III (JASCO, Japan), and with a multi-
wavelength detector MULTI-340 (JASCO,
Japan), respectively. The measurement with
MULTI-340 was carried out in order to obtain the
absorption spectra of the fractions over the wide
range of wavelengths, although the sensitivity of
the detector was less than that of the UVIDEC-
100-III detector.
Quantities of several retinoids were estimated
from their abosrption coefficients and the peak
areas of known amount of standard retinoids. The
fractions of 2-8 min (retention time) under our
HPLC conditions were used as the sample of
retinylester. The solvent was evaporated from the
fractions and the residue was incubated in 6%
KOH-methanol solution at 25°C for 1 hr for sapo-
nification. The amount of retinyl ester was calcu-
lated from that of retinol produced by the sapo-
nification.
Observation of fluorescent images of oil-droplets in
planarian tissues
The planarian worm of 5 mm in length was put
on non-fluorescent slide glass, covered with a thin
coverslip and spread by the squash method. The
fluorescent images of the oil-droplets in the spread
HPLC Analysis of Planarian Retinoids 943
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300 340 380 420
Wavelength (nm)
(a) HPLC profile of retinoids extracted from 18 bodies of planarian worms. (b) Absorption spectra of peak
fractions indicated by numbers 1 and 2. The worms were dark-adapted overnight in aged tap water at 20°C.
Extractions were carried out by the oxime methods.
specimen were observed using a flouorescence
microscope (Olympus inverted-microscope, IMT-
2, equipped with Olympus incident-illumination
type fluorescence apparatus, IMT2-RFL). The
Specimen was excited by light (50 W halogen lamp)
passing through an excitation-filter (UG-1) and
observed through a filter (L420) and a dichroic
mirror (DM 400). The photograph of fluorescent
images was taken using the color film (Fujichrome
DX 400D).
RESULTS
Retinoids detected in the planarian
Figure la shows an HPLC profile of the extract
of retinoids from the whole bodies of 18 planarian
worms of about 10 mm in length. This figure was
obtained by recording the absorbaces of the frac-
tions at 350 nm with the UVIDEC-100-III detec-
tor. Numbers 1 and 2 indicate peaks close to the
retention time of standard syn all-trans retinalox-
ime and all-trans-retinol, respectively. Two big
peaks between 1 and 2 are not identified. The
relative absorption spectra (300-420 nm) of the
fractions corresponding to peaks 1 and 2 were
obtained by the MULTI-340 detector, indicated as
curves 1 and 2 in Fig. 1b, respectively. The
absorption maxima of curves 1 and 2 clearly match
those of the standard syn all-trans retinaloxime
(Amax=358 nm) and all-trans retinol (Amax=325
Fic. 2.
planarian body. The specimen was obtained by the
squash methods. Bar=50 um.
Fluorescent micrograph of oil-droplets in the
nm), respectively. The fractions between 2 and 8
min, which contain retinyl esters, were collected
and saponified as described in Materials and
Methods. Then the material obtained after the
saponification was analyzed by HPLC. From these
analyses, the amounts of all-trans retinal, all-trans
retinol and all-trans retinyl ester were calculated as
2.5, 14.7 and 99.4 pmol/body, respectively.
Figure 2 shows fluorescent images of several
944 K. Azuma, N. Iwasaki et al.
oil-droplets in the planarian specimens obtained by
the suqash method. The light color of fluoresence
was green-yellow suggesting the presence of re-
tinol and/or retinyl ester. Probably, the planarian
worms store the all-trans retinyl ester in oil-
droplets, because the worms large
amounts of the retinyl esters, as mentioned above
(more than 85 mol% of total retinoids).
Figure 3 shows HPLC profiles of extracts from
the head (a) and the tail (b) pieces of 28 planar-
ians. Peaks numbered 1 and 2 are corresponding
to syn all-trans retinaloxime and all-trans retinol,
respectively, as estimated from their retention
times and abosorption spectra (data not shown).
Thus all-trans retinal and all-trans retinol were
found in the head and tail pieces of the planarian.
contain
D
2
A350
q 2-004
a con
10 min 10 min
Fic. 3. HPLC profile of retinoids extracted from both
the head (a) and tail (b) pieces of 28 planarian
bodies. The worms were dark-adapted overnight in
aged tap water at 20°C, separated into head and tail
pieces under dim red light and were extracted by the
oxime methods.
Retinals in the planarian head pieces
Figure 4a shows an HPLC profile of the extract
of retinoids from head pieces. The head pieces
were cut off from the 600 bodies of the planarian
under room light, then dark-adapted overnight in
aged tap water at about 20°C and stored at —20°C
until use. The chromatogram was obtained by
recording the absorbances of the fractions at 350
nm with the MULTI-340. Retention times of
a
0 10 20 30 40 50
Time (min)
Relative Absorbance
300 340 380 420
Wavelength (nm)
Fic. 4. (a) HPLC chromatogram of retinoids extracted
from head pieces of the planarian worms. (b)
Absorption spectra of peak fractions indicated by
numbers 1, 2 and 3. The planarian head pieces were
separated from 600 bopdies of the planarian worms
under room light, then dark-adapted overnight in
aged tap water at 20°C and stored at —20°C until
use. Extractions were carried out by the oxime
methods.
peaks 1, 2 and 3 are close to those of standard syn
11-cis retinaloxime, syn all-trans retinaloxime and
all-trans retinol, respectively. The relative absorp-
tion spectra of fractions corresponding to peaks 1,
2 and 3 are represented as curves 1, 2 and 3 in
Figure 4b respectively. Curves 2 and 3 are due to
the absorption spectra of syn all-trans retinaloxime
and all-trans retinol, respectively, as indicated in
Figure 1. Curve 1 seems to be corresponding to
the absorption spectrum of syn 11-cis retinaloxime,
because the shape of curve 1 is different from that
of surve 2 due to syn all-trans retinaloxime. The
amounts of all-trans retinal, 11-cis retinal and all-
trans retinol including the retinyl ester in different
preparations were ().1-1.1, 0.11-0.19 and 20-50
pmol/head, respectively.
In order to elucidate whether or not 11-cis and
HPLC Analysis of Planarian Retinoids 945
D
3 350
0.004
2
10 min
Relative Absorbance
oO
300 340 380 420
Wavelength (nm)
Fic.5. (a) HPLC chromatogram of hexane extract
from freeze-dried sample of the planarian heads
prepared as mentioned in Fig. 4. (b) HPLC chro-
matogram of retinoids extracted by the oxime
method from the residues after the hexane extrac-
tion. (c) Absorption spectra of peak fractions
indicated by numbers 2 and 3 (solid lines) and
standard syn 11-cis and syn all-trans retinaloximes
(dotted lines).
all-trans retinals were bound to any protein in the
planarian tissues, we carried out an experiment as
follows. The hexane extract from the freeze-dried
samples of planarian heads was analyzed by
HPLC. As shown in Figure 5a, the peak of all-
trans retinol (peak 1) was quite large, while the
peaks due to 11-cis and all-trans retinals were not
found. Figure 5b is an HPLC profile of retinoids
extracted from the residues by the oxime methods
after the hexane extraction. The figure indicates
substantial peaks, numbered 2 and 3, correspond-
ing to syn 11-cis retinaloxime and syn alll-trans
retinaloxime, respectively. Figure 5c indicates the
relative absorption spectra of fractions corres-
ponding to peaks 2 and 3 as curves 2 and 3. These
spectra are very similar to those of standard syn
11-cis retinaloxime and syn all-trans retinaloxime
(indicated by dotted lines), respectively. The early
fractions (2-8 min) in Figure 5a have peaks which
are much larger than those in Figure 5b, indicating
that retinyl esters were mostly extracted by the
hexane extraction. Thus hexane extracted almost
all of the all-trans retinol along with retinyl esters
in the planarian tissues leaving 11-cis and all-trans
retinals.
DISCUSSION
As shown in Figure 1, retinoids extracted from
the homogenates of 18 bodies of the planarian
were composed of all-trans retinal (2.1 mol%),
all-trans retinol (12.6 mol%) and all-trans retinyl
ester (85.2 mol%). Retinyl ester is probably a
main storage form of retinoids in the planarian
body and seems to exist in the oil-droplets, which
emit the green-yellow fluorescence as seen in
Figure 2. It has long been known that vitamin A is
stored mainly as retinyl ester in the livers of a
numbers of vertebrate species and that these re-
tinyl esters are present in oil-droplets of the liver
fat-storing cells [16]. The planarian has oil-
droplets in the fixed parenchymal cells [17]. Prob-
ably the planarian is capable of storing retinol in
ester form in the parenchymal cells.
The hexane extract from the freeze-dried planar-
ian heads contained almost all of the all-trans
retinol and the retinyl ester in the tissues. How-
ever, all-trans and 11-cis retinals could not be
detected in the extract. Both of the retinals were
extracted from the residues, after the hexane ex-
traction, as the oximes. It is well known that
retinals combined with amino group of a protein in
tissues (e.g. vertebrate and invertebrate retinas)
are not extracted by hexane. Therefore the retin-
als in the planarian may be bound to an uniden-
tified proteins in the tissues.
All-trans retinal was detected in extracts of both
head and tail of 28 planarians (see Fig. 3), while
11-cis retinal was detected in extracts derived from
600 planarian head pieces (see Fig. 4 and Fig. 5).
It is reasonable to infer that the 11-cis retinal was
derived from the chromophore of visual pigment
of the planarian photoreceptor. The eye of the
planarian used in this experiment is assumed to be
a sphere of about 90 4m in diameter. The rhodop-
sin concentration of invertebrate photoreceptors is
946
0.3-0.4 mM [18]. We can estimate the amount of
the chromophore per planarian eye, if the eye is
assumed to be filled with microvilli which contain
rhodopsin in the concentrations of 0.3-0.4 mM.
The calculated value of the amount of chromo-
phore was 0.11-0.15 pmol per eye, which was
close to the amount of 11-cis retinal indicated in
this experiment i.e., 0.11-0.19 pmol per head.
ACKNOWLEDGMENTS
The authors thank professor Dr. T. P. Williams (Flor-
ida State University) for critical reading of the manu-
script and Messrs. H. Fujino, T. Tanaka, A. Yuda and
M. Miyazaki for collections of the planarian worms.
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1 Tamamaki, N. (1990) Evidence for the phagocytotic
removal of photoreceptive membrane by pigment
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2 Brown, H. M. and Ogden, T. E. (1968) The
electrical response of the planarian ocellus. J. gen.
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3 Menzel, R. (1979) Spectral sensitivity and colour
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5 Fujita, J., Sakurai, N. and Shinozawa, T. (1991)
Presence of rhodopsin-like proteins in the planarian
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6 Knowles, A. and Dartnall, H. J. A. (1977) Habitat,
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Suzuki, T. and Makino-Tasaka, M. (1983) Analysis
of retinal and 3-dehydroretinal in the retina by
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Azuma, M. and Azuma, K. (1988) Retinoid
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Biosynthesis, absorption, and hepatic metabolism of
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Ishida, S. (1987) “Biology of planarians.” Ed. by W.
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ZOOLOGICAL SCIENCE 9: 947-954 (1992)
Effect of Disuse on Muscle Energy Metabolism Studied by
in vivo 31-Phosphorus Magnetic Resonance Spectroscopy
SHoy] FUKUTA, TAKAAKI IkATA and Iwao Miura!
Department of Orthopaedic Surgery, School of Medicine,
the University of Tokushima, 3-18-15, Kuramoto-cho,
Tokushima city, Tokushima 770, Japan
ABSTRACT— Chronic unloading induces disuse atrophy in rat hindlimb muscles. The purpose of this
study was to examine the effect of hindlimb suspension on energy metabolism in hindlimb muscles
during tetanic contraction using 31-phosphorus magnetic resonance spectroscopy (*1P-MRS). Muscular
contraction was induced by electrical stimulation of the sciatic nerve at 40 Hz. Energy metabolism was
evaluated by measuring the relative intensities of phosphocreatine (PCr), inorganic phosphate (Pi),
B-ATP and phosphomonoester (PME). The intracellular pH was also estimated from the chemical shift
of Pi. Two weeks of hindlimb suspension induced a greater weight loss in the soleus compared with the
gastrocnemius muscle. The maximum tension at 40 Hz was significantly lower (P< 0.05) in the hindlimb
suspended group than in the control group. However, fatigability did not differ significantly. The
energy level at rest was similar in the two groups. During electrical stimulation, the Pi/(Pi+ PCr) ratio
was higher in the hindlimb suspended group compared with the control group significantly (P<0.01),
but the intracellular pH did not differ. These results suggest that the metabolic capacity of hindlimb
muscle decreases after a prolonged period of disuse.
© 1992 Zoological Society of Japan
INTRODUCTION
Disuse atrophy of skeletal muscle has been
studied in several experimental models, including
small cage restraint [1], spinal cord transection [2],
denervation [3, 4], tenotomy [3, 5], cast immobi-
lization [6, 7], joint fixation [8] or paralysis with
tetrodotoxin [9]. In 1979, the hindlimb suspension
model was developed by Morey to simulate the
influence of weightlessness [10]. With the hind-
limb suspension model, in contrast to previous
models, the hindlimbs are made completely non-
weight bearing and the animal is able to both
contract and relax the hindlimb muscle freely.
Numerous investigators have begun to use this
model to examine the effect of unloading on
skeletal muscle and study the mechanism by which
disuse atrophy is undergone.
The changes which occur in the suspended hind-
Accepted July 22, 1992
Received March 19, 1992
' Bioenergetics Res. Center, Tokushima Inst., Otsuka
Pharmaceutical Co., Ltd. 463-10, Kagasuno, Kawau-
chi-cho, Tokushima city, Tokushima 771-01, Japan
limb have been studied histochemically. The per-
centage of slow-twitch fibers decreases in the
soleus after the hindlimb is suspended [11, 12],
while no such changes was observed in the gastroc-
nemius, tibialis anterior [13], and extensor digitor-
um longus muscles [12]. Significant changes in
contractile properties, therefore, may be due to
changes in the fiber composition of an unloaded
muscle.
Histochemical and biochemical enzyme assays
have shown that both anaerobic and aerobic capac-
ity increase in slow-twitch muscle [11-13] while the
aerobic capacity in fast-twitch muscles decreases
during hindlimb suspension [13]. These studies
were based on measurements made in vitro of
isolated muscles. However, it is best to measure
the enzymatic adaptation which affects muscle
energy metabolism during contraction in vivo.
31-phosphorus magnetic resonance spectroscopy
is being used to study the energy metabolism and
mitochondrial function of skeletal muscle [14, 15].
This technique permits non-invasive and repetitive
measurement of high energy phosphate, inorganic
phosphate, and sugar phosphate level, as well as
948 S. Fuxata, T. IkATA AND I. Miura
estimation of the intracellular pH. The purpose of
this study was to examine the effect of prolonged
muscle unloading on energy metabolism in vivo
during muscular contraction.
MATERIALS AND METHODS
Animal care Male Wistar Kyoto rats (20-24
weeks) were used in this study. Temperature
(25°C) and light-dark cycles (12:12 h) were main-
tained throughout the experimental period. Ex-
perimental rats were divided randomly into the
control and hindlimb suspended (HS) groups.
Hindlimb suspension procedure The suspen-
sion method used in this study was a modification
of Morey’s tail-suspension model [10]. After the
rat was anesthetized with pentobarbital sodium (50
mg/kg body weight), 1.2 mm diameter holes were
drilled carefully through the third and fourth tail
bones without disturbing blood flow to the tail. A
stainless steel wire was inserted and attached to the
top of the cage by a ball-bearing wheel swivel
which allows 360° rotation. The suspension height
was adjusted to prevent the hindlimbs from con-
tacting the floor or the sides of the cage while the
forelimbs were allowed to support their weight and
to obtain food and water ad libitum.
Experimental preparation and muscle
stimulation After 2 weeks of suspension, the
rat was anesthetized with pentobarbital sodium (50
mg/kg body weight). The right sciatic nerve was
exposed in the gluteal region and placed in contact
with a small bipolar electrode. The right hindlimb
was placed in a 2cm diameter solenoid coil and
was fixed on a small platform at both the knee and
ankle with full extended position. The distal
tendon of the gastrocmenius-plantaris-soleus mus-
cle group was exposed at the ankle, cut, and
attached to a strain gauge (TB611, NIHON
KOHDEN) with a noncompliant thread, the out-
put of which was displayed on a Polygraphy system
(RM-6000, NIHON KOHDEN). The rat was
inserted in the magnet. The hindlimb muscles
were stimulated electrically (SEN-3301, NIHON
KOHDEN) through an electrode with a 0.5 ms
square wave pulse at 40 Hz for 20 min. Stimula-
tion was performed with intermittent trains of 1
sec in duration, repeated every 2 sec. Stimulation
voltage was 4 V which was supramaximal in both
the control and HS groups.
31-phosphorus magnetic resonance spectroscopy
(“P-MRS) _*'P-MRS spectra were recorded
with a BEM 250/80 spectrometer (OTSUKA
ELECTRONICS U.S.A. INC.) operating at 32.3
MHz. The spectrometer was operated in the
Fourier transform mode with a pulse duration of
15 ws (90°) and a pulse interval of 2sec. Each
spectrum was averaged over 2 min, which was
from 60 free induction decays (FIDs). The phos-
phocreatine peak (PCr) was assigned as a chemical
shift of zero. The levels of tissue PCr, inorganic
phosphate (Pi), @-ATP and phosphomonoester
(PME) were estimated from the areas under indi-
vidual peaks. The cellular energy level was evalu-
ated by the Pi/(Pi+PCr) ratio and the relative
concentration of Pi, PCr, ATP, and PME were
calculated as fractions of the total 7'P-MRS signal.
Pulse interval of 2 sec reduced the PCr and Pi
amplitudes to about 60% of their respective maxi-
mum. These saturation factor were incorporated
into the calculation of relative concentration. The
intracellular pH was estimated from the chemical
shift of Pi relative to the PCr peak (6) and calcu-
lated as follows [16]:
Intracellular pH=6.90— |(6—6.81)/(3.29—6)|
Measurement of muscle weight At the end of
the experiment the rat was killed with an overdose
of pentobarbital sodium. The hindlimb muscles
were removed, cleaned of connective tissue, and
weighed. Weights were expressed in absolute (mg)
or relative (mg/g body weight).
Data analysis
+S.E. Differences were tested for significance by
Student’s f-test.
Values were expressed as mean
RESULTS
The absolute and relative muscle weights from
the control and the HS groups are shown in Table
1. They were significantly lower in the HS group
than in the control group. The muscle mass loss
*'P-MRS Study of Disuse Muscle Atrophy 949
TABLE 1.
body weight ratios
Effect of hindlimb suspension on body weight, muscle weight and muscle weight to
Control hindlimb suspension Yo change
(6) (6)
BW(g) 378.0+3.1 349.0+5.0 = 57°
MW(mg)
soleus 170.8+4.2 104.4+3.6 = 38,9"
plantaris 421.3+5.6 350.5 +4.0 =loi3""
gastrocnemius 1991.7+47.2 1557.6 +53.6 = ils."
MW/BW(mg/g)
soleus 0.448 + 0.009 0.297 +0.013 = 33.1"
plantaris 1.111+0.014 1.006 + 0.021 = 95°"
gastrocnemius 5.020 +0.136 4.462 +0.128 = iii
Values are means+S.E. The number of rats in each group is given in parentheses.
weight; MW, muscle weight; MW/BW, muscle weight to body weighg ratios.
BW, body
Significant differences
between control and hindlimb suspension groups are indicated by *(P<0.05) and ** (P<0.01).
TABLE 2. Effect of hindlimb suspension on contrac-
tile properties in the soleus-plantaris-gastro-
cnemius muscle group
control hindlimb suspension Ychange
(6)
P.(g) 413.6+5.9 336.5+13.7 —18.6*
P./BW 167.1+1.1 167.0+1.5
Values are means+S.E. The number of rats is
given in parentheses. Abbreviations: P,, maxi-
mum tension at 40Hz; BW, body weight. A
significant difference between control and hindlimb
Suspension is indicated by * (P<0.05).
100
80
% tension
Time
Fic. 1.
10 20
after the onset of stimulation
Time course of changes in the % tension during electrical stimulation. HS, hindlimb suspension.
was greater in the slow-twitch soleus than in the
predominantly fast-twitch plantaris and gastrocne-
mius muscles.
The maximum tension at 40 Hz in the control
and HS groups were 413.6+5.9 g and 336.6+13.7
g, respectively (Table 2). It was significantly lower
in the HS group when compared with the control
group (P<0.01). However the tension per gram of
muscle did not differ between the control and HS
groups. The percent decline in tension (% ten-
sion) 2 min after the onset of electrical stimulation
—c— Control
(min)
950, S. Fuxata, T. IKATA AND I. Miura
was 39.1+2.0% in the control group and 33.6+
2.3% in the HS group. At the end of the stimula-
tion % tensions were 21.3+0.2 and 21.1+0.2%,
respectively. There was no significant difference in
the % tension decline between the two groups
(Fig. 1).
The relative signal intensities of PCr, ATP, Pi,
PME and the intracellular pH did not differ signifi-
cantly at rest between the control and HS groups
(Table 3).
Typical *!P-MRS spectra taken at rest, during
electrical stimulation, and during recovery are
TABLE 3. Relative signal intensities at rest of phos-
phomonoester (PME), inorganic phosphate (Pi),
phosphocreatine (PCr), adenosine triphsphate
(ATP) and intracellular pH in control and hind-
limb suspension
control hindlimb suspension
(6)
PME 4.25+0.28 3.43 +0.41
Pi 4.03 +0.28 4.28+0.22
PCr 71.93 £0.45 72.42+0.39
B-ATP 20.67 £0.72 19.87+0.76
intracellular pH 7.08 £0.03 7.08 +0.03
Values are means+S.E. The number of rats is
given in parentheses.
Control
Fic. 2.
shown in Figure 2. Decreases in the PCr peak and
increases in the Pi peak were observed during
electrical stimulation. A lower level of PCr peak
and higher level of Pi peak were maintained in the
HS group when compared with the control group.
After 10 min of recovery, the Pi and PCr peaks in
both groups returned to the resting values.
The time course of changes in the Pi/(Pi+ PCr)
ratio is shown in Figure 3(A). In the first 2 min of
the stimulation, the Pi/(Pi+PCr) ratio in the
control and HS groups increased to 0.71 + 0.03 and
0.84+0.02, respectively. After that, they returned
gradually with time and were 0.55+0.04 and 0.74
+0.03 at the end of the stimulation. The Pi/(Pi+
PCr) ratio was significantly higher in the HS group
than in the control group during the stimulation.
During the first 8 min of recovery, the Pi/(Pi+
PCr) ratio in both groups decreased rapidly and
returned to the resting values without significant
difference.
The time course of changes in the intracellular
PH is shown in Figure 3(B). After the onset of the
stimulation, the pH decreased and reached its
lowest level within 2 min (6.36 +0.02 in the control
group and 6.45 +0.03 in the HS group, respective-
ly). From 2 min to the end of the stimulation, the
intracellular pH increased gradually and returned
A typical set of *'P-MRS spectra at rest, during stimulation, and during recovery. HS, hindlimb suspension;
PME, phosphomomoester; Pi, inorganic phosphate; and PCr, phosphocreatine.
IP_MRS Study of Disuse Muscle Atrophy 951
(A) : }
Rest Stimulation Recovery
Ale [-——=19} (Clojnilitpell|
X=---k HS
6)
&
a OF
a
40. (min)
Time after the onset of stimulation
(B) Rest Stimulation Recovery
Ht t 1
e—5 CON
X----x HS
ae
Q.
iS
2
oO
(3)
ee
ie
2 = 4 + + an 1
0 40 20 30 40 (mln)
Time after the onset of stimulation
Fic. 3. Time course of changes in the P1/(Pi+ PCr) ratio (A) and the intracellular pH (B) at rest, during stimulation,
and during recovery. Values are means+S.E. The number of rats in 6 in each group. HS, hindlimb suspension;
Pi, inorganic phosphate; and PCr, phosphocreatine. Significant differences between control and HS groups are
represented by * (P<0.05) and ** (P<0.01).
to the resting value within 10 min of recovery.
There was no significant difference in the intra- UISOUSISIROS
cellular pH between the two groups throughout Chronic unloading induces disuse atrophy in rat
the experimental period. hindlimb muscles but the degree of muscle atrophy
varies in which the slow-twitch muscle is more
952 S. FuKATA, T. IKATA AND I. Miura
susceptible to unloading that fast-twitch muscle.
In the present study, the decrease of wet weight
was greater for the soleus muscle than for the
gastrocnemius muscle. These results are consistent
with those of previous studies [13, 17-19].
A singificant decrease in the maximum tension
was demonstrated after 2 weeks of suspension.
The control and HS groups were similar with
respect to the tension developed per gram of
muscle. These results indicate that muscle atrophy
may occur without selective myofibrillar protein
loss during hindlimb suspension. The percent
decline in tension development did not differ sig-
nificantly between the two groups. In contrast,
Fell et al. [20] reported faster rates of fatigue in
gastrocnemius muscle after 1 week of suspension.
This discrepancy might be due to differences in the
stimulation parameters. Fitts and Hollozsy [21]
reported that the degree of fatigue measured dur-
ing a period of stimulation varied with the stimula-
tion parameters used for the measurements. The
stimulation parametes that we used induced con-
traction at a higher intensity than did those used by
Fell et al. In suspended rat the gastrocnemius
muscle, therefore, is thought to be more fatigable
during stimulation at a lower inensity.
Chronic unloading affects musclar energy state
as well as mechanical properties in the hindlimb
muscle. Biochemical study of high energy phos-
phate concentrations in atrophied muscles re-
ported that the concentrations of PCr and ATP
were unchanged in the soleus while a significant
reduction in ATP concentrations was noted in the
extensor digitorum longus muscle [12]. In con-
trast, Jaspers et al. [24] reported that the ATP
concentration increases in the soleus muscle after 6
days of tail-cast suspension, although they did not
examine that in fast-twitch muscles. However, the
ATP concentration relative to total adenosine
nucleotide was unchanged in both the soleus and
the extensor digitorum longus muscle [12]. In this
study, relative concentrations of high energy phos-
phates were calculated for the estimation of energy
state. At rest, high energy phosphates did not
differ significantly between the control and HS
groups. This result agrees with in vitro assay.
There are some *'P-MRS studies of energy levels
in skeletal muscle during disuse. Zochodne et al.
[22] reported that the patients with denervated
muscles had a lower PCr concentration and a
higher intracellular pH than did healthy control
resting muscles. In contrast, the patients who had
cast immobilization did not exhibit these changes.
This difference is likely due to the type of disuse.
Denervation removes trophic neural influence, by
which the metabolic properties in skeletal may be
affected.
High energy phosphates during electrical stimu-
lation was significantly lower in the HS group than
in the control group, whereas no difference was
observed in the intracellular pH during electrical
stimulation. Similar phenomena were observed in
the patients with mitochondrial myopathy [23, 24].
In the patients with mitochondrial myopathy, the
PCr concentration decreases to low values during
exercise but without an accompaning severe in-
tracellular acidosis in contrast to the observation in
Since free ADP is believed to be the
principal driving force of mitochondrial respiration
in skeletal muscle, the increase Pi concentration
and the decreased PCr concentration suggest that
there exists the elevated ADP concentration due
to poor coupled oxidative phosphorylation in sus-
pended hindlimb muscle. These results suggest
that the decrease in aerobic capacity after hindlimb
suspension induced low energy level during elec-
trical stimulation.
The enzymatic adaptation of rat hindlimb
muscle after hindlimb suspension has been re-
ported [11, 13]. It is suggested that the capacity for
glycolysis increases in slow-twitch and fast-twitch
oxidative glycolytic fibers while fast-twitch glyco-
lytic fibers are affected minimally. Aerobic capac-
ity is reported to increase in slow-twitch fibers but
decreases in fast twitch fibers. The result obtained
by *'P-MRS are consistent with the enzymatic
changes in fast-twitch fibers during hindlimb sus-
pension.
However, enzyme activities may not be the only
factor to muscular energy metabolism in vivo.
Oxygen delivery to the hindlimb muscle also
affects the energy metabolism during musclar con-
tractions. Since the ratio of capillaries per fiber in
the soleus decreases 46% [12], oxygen delivery
may also decrease. Sogabe et al. [25] reported that
controls.
low inspired oxygen caused low energy level dur-
‘IP-MRS Study of Disuse Muscle Atrophy
ing muscular contraction without intracellular
acidosis.
Both changes in enzyme activites and
oxygen delivery may induce lower energy state in
suspended hindlimb muscle during contraction.
While further study is required to assess the
mechanism of metabolic adaptation during unload-
ing of the hindlimb by the suspension, the present
study demonstrates that *'P-MRS may be used to
assess the biochemical changes which occur in
skeletal muscle under a variety of pathological
conditions.
10
11
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Jaspers, S. R. and Tischler, M. E. (1984) Atrophy
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25 Sogabe, T., Tsubo, M. and Miura, I. (1989) Effect
ZOOLOGICAL SCIENCE 9: 955-969 (1992)
© 1992 Zoological Society of Japan
Initiation of Spermatophore Protrusion Behavior in the
Male Cricket Gryllus bimaculatus DeGeer
TAKAHIRO OotsuBo and MASAKI SAKAI’
Department of Biology, Faculty of Science, Okayama University,
Tsushima-Naka-3-1-1, Okayama 700, Japan
ABSTRACT—Spermatophore protrusion is the reproductive act of the male cricket ejecting the
immature spermatophore to the external genitalia to solidify.
Forty-five seconds prior to this act
spermatophore materials are secreted into the ejaculatory duct. Initiation of spermatophore protrusion
was facilitated by contact with the female: the interval between the end of copulation and sperma-
tophore protrusion was considerably shorter in the male with the female (6 min) compared to the male
with no female (>60 min) after copulation. It was also accelerated to a similar extent through increased
sensitivity to the female when males were deprived of females for at least 12-24 hr prior to testing. On
the other hand, the occurrence of spermatophore protrusion was delayed or prevented when the male
was restrained in an abnormal posture. Spermatophore protrusion still occurred, though more slowly,
in males with the connectives transected between the thorax and the abdomen.
INTRODUCTION
During copulation, the male insect gives the
female a spermatophore, a proteinous capsule
containing sperm. In the cricket Gryllus bimacula-
tus, the male starts tc prepare a new sperma-
tophore for the next mating about 6 min after
copulation [1]: the white milky mass is ejected
from the genital cavity into a space between the
ventral lobes of the external genitalia. This act is
called spermatophore protrusion. Once it occurs
the male begins to sing a calling song at a fixed
interval [2-5]. Thus, spermatophore protrusion is
taken as the first step in the sequence of the male
cycle.
Although the structure and formation of sper-
matophores of insects have been studied extensive-
ly [6-8], the initiation mechanism of sperma-
tophore protrusion is not yet known. Behavioral
study indicates that the female does not play a role
in stimulating the initiation of spermatophore
protrusion [3]. Our previous study showed that the
interval (CPPT) between the end of copulation
Accepted July 23, 1992
Received May 23, 1992 J
' To whom reprint requests should be addressed.
and spermatophore protrusion varied among
males [5]. CPPT was extremely long in some males
[5], and considerably prolonged by adverse stim-
ulation of the genitalia [1, 4]. These suggest that
the process of spermatophore protrusion may be
affected by some external and internal factors
which is different from the process of the calling
song starting with a constant latency after sperma-
tophore protrusion [2-5]. On the other hand,
physiological study indicates that communication
between the brain and the terminal abdominal
ganglion was indispensable for spermatophore
protrusion [9]. In particular, the pars intercerebra-
lis of the brain seemed to be essential because its
coagulation abolished this behavior [10]. There is,
however, a conflicting result that elimination of the
corpora allata or the corpus cardiacum, which is
the terminal regions of the projection neurons in
the pars intercerebralis [11], failed to abolish sper-
matophore protrusion [2].
Thus, we investigated external and internal fac-
tors that may affect the initiation of sperma-
tophore protrusion and estimated nervous com-
munication between the brain and the terminal
abdominal ganglion.
956 T. OorsuBo AND M. SAKAI
MATERIALS AND METHODS
Animals
Crickets Gryllus bimaculatus DeGeer reared at
a 12L:12D photoperiod at 27+2°C were used.
We previously reported CPPT varied considerably
among individuals reared alone and gradually de-
clined as males matured [5]. In the present study,
we used males reared in a group because a large
number of insects were used for various experi-
ments. The daily change in CPPT (Fig. 1A) was
checked in males (n=30) reared in a group with
females (n=60) in a container (45 x 30X25 cm).
CPPT was much longer and more variable, as
compared with that in males reared individually
isolated (Fig. 1B) and those (n=30) reared in a
group with males (Fig. 1C) for the first 6 days after
60
aN
(e)
= nN
{e) {e) (e)
Median of CPPT (min)
ine)
[e)
molt and then reached a constant level after day 8.
Thus, crickets aged 8-10 days after molt were
used. Males reared in the two latter conditions (B
and C) were not used here except in only one
experiment.
Behavior observation and data analysis
The male and female were paired in a 200 ml
beaker. Behavioral events were checked by eye,
but antennal and abdominal movements were re-
corded on video tape and later analyzed by slow
speed play back [1]. After they copulated once,
the time of reproductive events, i.e. the pairing of
male and female, beginning of the calling song,
spermatophore extrusion during copulation, the
opening of the subgenital plate and spermatophore
protrusion was recorded with a stop watch. In the
present experiments, the end of copulation was
W
Ee
E
kK
o
OS tS aida MC 8 10
°
Ae
z [| 8
10
a rr
ois 4 5 6 8 10
Days after imaginal molt
Fic. 1.
Developmental changes in CPPT of spermatophore protrusion in males reared under 3 different conditions
after molt. A: males reared in a group with females. B: males reared alone. C: males reared in a group with
males. Each experiment started with 30 males but the number reduced to 29 in A and C, and 24 in B on day 10th
owing to death.
Each point represents the median value of CPPT and each bar, the 95% confidence interval
(C.I.). The dotted line in A indicates the | hr observation time limit. Some of the same conventions are adopted
in the subsequent figures.
Spermatophore Protrusion in Crickets 957
defined as the moment of genitalia separation (GS
in Fig. 2) which regularly occurred 10sec after
spermatophore extrusion [1], i.e., extrusion of the
attachment plate of the spermatophore from the
dorsal pouch [1]. Spermatophore protrusion was
defined as the moment of ejection of an immature
spermatophore from the genital cavity (SPPT in
Fig. 2). Thus, CPPT is represented as the interval
between GS and SPPT. CPPT was measured in
minutes rounding off fractions to one decimal
place. Data were expressed as medians with 95%
confidence intervals (C.I.). Statistical significance
on CPPT was set at P<0.05 by the Mann-Whitney
U test unless otherwise stated.
Separation from the female
To examine the effect of the female on CPPT,
two different procedures were used.
During _ testing: When copulation
accomplished, the female was removed from the
beaker (“male” condition) and CPPT was timed.
For comparison, the female was replaced with a
male (“male and male” condition). Control was
the “male and female” condition in which the male
and female were simply left after copulation.
Was
Prior to testing :
ly removed from the container before experiments
and males experienced female-deprivation for 4, 8,
12, 24 or 72 hr prior to pairing. According to Fig.
1C, CPPT was expected to be shortened by these
treatment. They were tested in the “male” condi-
tion. The reason for this will be described later.
Modifications of these procedures will be de-
scribed in the places concerned.
All females were temporari-
Restraint of males
In order to examine whether spermatophore
protrusion could occur in abnormal conditions,
males were restrained in four different conditions
immediately after copulation (Table 2). 1) Posture
normal, legs contacted; the male was glued by the
dorsal pronotum to a metal bar in a normal posture
and placed on a movable styrol ball. 2) Posture
normal, legs restrained; each tarsus of the 6 legs
was fixed to the substrate with a piece of Scotch
tape. 3) Posture reversed, legs contacted; the male
was glued by the dorsal pronotum to a wooden
mound with the ventral side up. A wooden plate
was set over the male to contact with the legs on it.
4) Posture reversed, legs suspended; the same as in
3) except for no plate. All the males were given a
periodic brushing of their antennae with female’s
antennae for 2—3sec every 40sec as female sti-
mulation. If the male did not show spermatophore
protrusion within a | hr observation period, it was
checked again after 24 hr during which they were
left without female stimulation.
Inspection of the spermatophore in the ejaculatory
duct
The ejaculatory duct was examined for an imma-
ture spermatophore in males at 4 different phases
of the male cycle (Table 1); after spermatophore
extrusion in copulation, after subgenital plate
opening, after spermatophore protrusion, and an
arbitrary time after the start of courtship before
copulation. Dissection was made soon after freez-
ing the males in liquid nitrogen (—192°C).
Ablation of sensory organs and reproductive organs
To block visual input, the compound eyes and
oscelli were covered by black lacquer. To reduce
chemical input, the antennae, palpi and tarsi were
cut off with scissors. For reproductive organs, the
accessory glands or the testes were removed with a
pair of forceps after opening the cuticles of the
respective abdominal segments. This was done on
day 3 after molt for the males to recover sufficient-
ly before testing.
Transection of the nervous system
After the male copulated, it was anesthesized by
cooling at 4°C, fixed on a cork board, and the
cuticles over the intended level of the connectives
or brain were cut (Fig. 9 inset). The connectives
were cut at different levels; between the brain and
the suboesophageal ganglion (Fig. 9e), between
the metathoracic ganglion and the 1st abdominal
ganglion (Fig. 9c), and between the 4th abdominal
ganglion and the 5th abdominal ganglion (Fig. 9b).
The connectives between the suboesophageal
ganglion and the prothoracic ganglion was sepa-
rated by cutting off the head at the neck (decapita-
tion Fig. 9d). The brain was cut 3 ways with a
958 T. OorsuBo AND M. SAKAI
razor blade; cut horizontally to separate the anter-
ior region (Fig. 9f), cut vertically at the midline to
separate it into the left and right (Fig. 9j), or cut
diagonally to separate the antero-lateral regions
(Fig. 9h) or the antero-median region including
the pars intercerebralis (Fig. 9g). The corpora
allata and the corpus cardiacum were removed
together with forceps (Fig. 91). Control males
were only anesthetized for the same duration as
transected males. All tests started on the day of
surgery.
Spermatophore checking in nervous system tran-
sected males
To examine the operated male’s ability to pro-
duce the spermatophore, the interior of the sub-
genital plate was checked every 6 hr for 3 succes-
sive days during which each male was kept isolated
in a 200 ml beaker. A spermatophore was marked
by India ink to discriminate from a new one. The
number of males that produced spermatophore(s)
during the 3 days, and the number of sperma-
tophore(s) produced per male per day were
counted.
RESULTS
Process to spermatophore protrusion
Behavior
Main reproductive events are shown in abbre-
viations in the middle of Fig. 2. The male copu-
lates with the female some time after pairing
(MFP). When the genitalia separated after sper-
matophore extrusion (GS in Fig. 2), all bodily
movements stopped. In 1 sec (C.I. 1-2, n=21), a
half (51%, n=41) of the males, then, exhibited
post-copulatory body thrust [5] similar to copula-
tory movement for 18sec (C.I. 14-30, n=21).
These movements occasionally continued for up to
a few minutes interposed by short rests. It finally
ceased when the male lowered its abdomen itself
(71%, n=21) but, in the remaining 29%, the body
thrust was terminated by the female’s movements
on the male’s back. Then, the male crept forward
from under the female with his body rocking in a
typically aggressive expression (MEFS in Fig. 2). A
distribution of the time interval between genitalia
separation (GS) and male-female separation
(MES) is shown in Fig. 2A. The GS-MSF interval
distributed from 5 to 80 sec with a median value of
18sec (C.J. 8-23, n=29). The interval (30sec
(C.I. 6-43, n=21)) of males exhibiting body thrust
was significantly longer than that (8 sec (C.I. 4-12,
n=20)) of those not exhibiting body thrust. This
post-copulatory body thrust developmentally
emerged on day 4th after molt [5] and may play a
role in keeping the female quiet longer to secure
the settlement of the spermatophore in the genital
chamber of the female.
After male-female separation, the male exhi-
bited watching behavior [12] or guarding behavior
[3]. Occasionally two kinds of characteristic move-
ments were observed; one, abdominal contraction
similar to those seen in evacuation of feces in a
strained posture and the other, rapid antennal
vibration at 10 Hz in the antero-postero direction
[13]. One example of the occurrences of these
movements in the male is shown by bars on the two
lines in the middle of the Figure 2. The frequency
of the abdominal contraction reached a maximum
around spermatophore protrusion. It may reflect
the activity of the reproductive organs to prepare a
new spermatophore or warm-up for its ejection.
Shortly before the spermatophore was ejected, the
male often repeated 2-3 small openings of the
subgenital plate. Then, the plate was widely pulled
down (Fig. 2B inset) referred to here as subgenital
plate opening (SGPO). The interval between
male-female separation (MFS) and subgenital
plate opening (SGPO) was 4 min (C.I. 3-9, n=29,
Fig. 2B).
Then, the abdominal contraction with the back-
ward pushing of the genitalia complex became
frequent and the immature spermatophore was
ejected through the genital opening to the space
between the ventral lobes, that is, the moment of
spermatophore protrusion (Fig. 2C inset). The
interval between subgenital plate opening (SGPO)
and spermatophore protrusion (SPPT) was 45 sec
(C.I. 43-49, n=28), very constant for its shortness
with a small C.I. (Fig. 2C).
Timing of spermatophore materials secretion into
the ejaculatory duct
Prior to spermatophore protrusion, the mate-
Spermatophore Protrusion in Crickets
B
mc
x M=4min Mes
© 40 ey
SE
@o
{=
5
2 20
(Ss)
iS
0 Jen
Oo Lo ao Gogo
Interval (min)
Ant. movement
Abd. movement A AA A
MFP GS MFS SGPO SPPT =
A 6Of MN
Occurrence (%)
Interval (sec)
959
60
| as M=45sec
: 5
an
O
20r
Occurrence (%)
0 2 40 GO
Interval (sec)
Fic. 2. Reproductive events and time intervals between them. In the center, the occurrences of antennal (upper)
and abdominal (lower) movements shown by vertical bars. The male was paired with a female at MFP. Scale bar,
10 sec. A-C: histograms of the time intervals. Time bin, 5 sec in A and C, and5 minin B. A: distribution of the
time interval between genitalia separation (GS) and male-female separation (MFS). Solid line, males (n=20) not
exhibiting post-copulatory body thrust. Dotted line, males (n=21) exhibiting post-copulatory body thrust. B:
distribution of the time interval (n=29) between MFS and subgenital plate opening (SGPO). C: distribution of
the time interval (n=28) between SGPO and spermatophore protrusion (SPPT). The histogram representing
>60 min in B indicates males which failed to exhibit SGPO within the time limit. Some of these conventions are
adopted in Fig. 4. M, median value; S, spermatophore; SGP, subgenital plate; VL, ventral lobes.
rials of the spermatophore are secreted from the
testes and accessory glands into the ejaculatory
duct. To determine this timing, the inside of the
ejaculatory duct was examined using males quickly
frozen at different phases in the male cycle. The
spermatophore was not found in any phase except
the very short period between the subgenital plate
opening and spermatophore protrusion (Table 1).
This means that spermatophore formation is initi-
ated at the moment of subgenital plate opening.
Some critical observations are shown in Fig. 3.
The ejeculatory duct was vacant in males in the
period between the end of copulation and sub-
genital plate opening (Fig. 3A). Ten seconds after
subgenital plate opening, a white mass containing
spermatozoa and the pressure body are seen (Fig.
3B). Thirty seconds after subgenital plate open-
ing, the spermatophore moves toward the genital
opening (Fig. 3C).
We observed that a white mass emerged from
the accessory glands into the vacant ejaculatory
duct in the male in phases other than the one
between subgenital plate opening and sperma-
tophore protrusion when the dissected male was
left to thaw at room temperature. This curious
phenomenon seems to be an artifact caused by a
960 T. OorsuBo AND M. SAKAI
TABLE 1. Presence or absence of the spermatophore in the ejaculatory duct and ventral lobes
Time after reproductive events Ejaculatory duct Ventral lobes n
Spermatophore 0 x x 10
extrusion 10 (min) x x 5
30 x x 5
Subgenital plate 0 @©5 Xx x 10
opening 10 (sec) O x 10
30 © x 5
Spermatophore 0 x O 10
protrusion 10 (min) x O 5
30 x O 5
Calling song >0 (min) x O 10
x absence of spermatophore. © presence of spermatophore. ©, x presence of spermatophore in
only 4 males. n, number of males used.
A pre-SGPO B post-—SGPOt0 c post—SGPO30
Fic. 3. Timing of the emergence of the spermatophore in the ejaculatory duct. Insects were frozen at 3 different
phases in the male cycle. A: between the end of copulation and subgenital plate opening (SGPO). B: 10sec after
SGPO. C: 30sec after SGPO. AG, accessory glands; DP, dorsal pouch; ED, ejaculatory duct; GO genital
opening; PB, pressure body; Sz, spermatozoa; VL, ventral lobes. Scale bar, 1 mm.
loosening of the accessory glands due to tissue role in intiation of spermatophore protrusion,
destruction. CPPT was measured in 3 different conditions (see
' methods); “male and female” (Fig. 4A), “male
Role of the female in spermatophore protrusion and male” (Fig. 4B) and “male” (Fig. 4C). In A as
a control, 82% (n=32) of the males showed sper-
Removal of the female during testing matophore protrusion within 10 min. The median
To examine whether the female plays a major — of CPPT was 6 min (C.I. 5—8) with 3 min being the
Spermatophore Protrusion in Crickets 961
LA +2 .B G+S .C &
M=6min M=>60min M=>6O0min
60r #l 60r
r L
%
w 40} 4o} a
oO
‘S
<b)
mae | | L
=)
(o)
[S)
S
20 20 20
L | L
o) 30 60 560 O 30 —60 s60 9 30 60 >60
CPPT (min) CPPT (min) CPPT (min)
Fic. 4. Effect of the female presence after copulation on CPPT. A: control (“male and female”, n=32). B: the male
paired with a male after copulation (“male and male”, n=26). C: the male separated from the female after
copulation (“male”, n=36). Time bin, 5 min.
shortest. In contrast, the male paired with a male
after copulation made spermatophore protrusion
in only 31% (n=26) within 1 hr (B). They were
often fighting at the beginning. No males showed a
CPPT shorter than 10 min. In C, 42% of males (n
=36), which were kept quiet with no female after
copulation, made spermatophore protrusion with a
CPPT of >60 min (C.J. 12->60). Twenty five
percent of them showed a CPPT shorter than 10
min. There was a significant difference between B
and C in the number of males exhibiting sperma-
tophore protrusion within 1 hr. In B and C, males
tend to split into two groups: those exhibiting
spermatophore protrusion within half an hour and
those not exhibiting it within 1 hr. In sum, these
results reveal that spermatophore protrusion is
considerably facilitated by the presence of the
female.
Then, the effect of the female’s presnece on
CPPT was examined by changing the duration of
pairing after copulation. Figure 5 shows the re-
sults. CPPT did not significantly decrease until the
duration of the female presence was prolonged to 5
Median of CPPT (min)
Fic.
Duration of female presence
after copulation (min)
5. Time dependence of female presence on CPPT.
The abscissa represents the duration (min) of the
presence of the female after copulation in the beak-
er. Asterisk, the male paired with a female again for
only 1 min between 4 and 5 min after copulation.
Each point represents the median for 30 males.
962 T. OorsuBo AND M. SAKAI
min (C.I. 5-19, n=30). However, this may be due
to the change in male’s internal state starting
several min after copulation. Yo examine this
possibility, the male was paired with a female for
only 1 min between 4 and 5 min after copulation
(asterisk). This short presence of the female was
not sufficient to facilitate the initiation of sperma-
tophore protrusion as seen in the CPPT of 41 min
(C.I. 18->60, n=30).
Block of sensory input
To study the nature of female stimulation, visual
or chemical input was eliminated. CPPT in blind
males (E in Fig. 6A) was 5.5 min (C.I. 3-7, n=20)
which was not significantly different from that of
intact males (Int in Fig. 6A). However, CPPT in
A
60 eee c cece cece cccccccccccccscccccccs 60
e
S 40
Fk
ae
OW
O
==
(@)
=
2 2O
n@)
®
=
Int E
Fic. 6.
=20) whose compound eyes and oscelli were covered with black lacquer.
A,P&T
Analysis of female factors. A: the effect of ablation of sensory organs. Int, intact (n=32). E, blind males (n
males with antennae, palpi and tarsi removed (A,
P & T in Fig. 6A) was 12 min (C.I. 7-23, n=25)
which was much shorter than that of males tested
without a female (Fig. 4C) but significantly longer
than that of the intact males. This means the
shortening of CPPT still occurred via male’s
chemoreceptors on the body surface as well as
those on the antennae, palpi and tarsi. To deter-
mine whether receptors were olfactory or taste,
the female was suspended by a string 8 cm over the
male without contact (non-C in Fig. 6B). CPPT
was >60 min (C.I. 18->60, n=28). For control,
a female was suspended 3cm above the male
allowing contact with her body. CPPT was 10.5
min (C.J. 5-18, n=20) which was much shorter
than that of the non-C group and not significantly
B
fe)
nonc C
A, P & T, males (n=25) whose
antenna, palpi and tarsi were removed. Note CPPT in A, P & T is much shorter than that of non-C, but still
significantly longer than that of the Int. B: the effect of contact with the female. non-C, males (n=28) which
could not contact with the female.
C: males (n=20) which could contact with the female.
A female was
suspended 8 cm (non-C) and 3 cm (C) above the male, respectively.
Spermatophore Protrusion in Crickets 963
60 Cece cc ccccccccccceccccccesccs
pS
oO
pe)
O
Median of CPPT (min)
Pee ecrescceseseseseseseseesesssessoesseoseceoes
Lendl
o" Cone lame: aE Sapo aT 172
Duration of deprivation (hr)
Fic. 7.
Effect of female-deprivation prior to testing on CPPT. The abscissa represents the time (hr) during which
males (n=30) had been separated from females prior to testing. Note the long latency at 0, 4, and 8 hr is
characteristic of males tested in the “male” condition as in Fig. 4C.
longer than that of the intact group (Fig. 6A), This
result reveals that female stimulation is not olfac-
tory, but presumably contact-mediated.
Female-deprivation prior to testing
Males were temporarily separated from the
females for a fixed time prior to pairing since
CPPT was shorter in female-deprived males during
the sexual maturation process after molt (Fig. 1B,
C). During testing, CPPT was measured in the
“male” condition so that the results would be much
clearer than in the “male and female” condition
because CPPT was expected to be shortened by
deprivation (compare Fig. 4A with Fig. 4C). As
shown in Fig. 7, the control (Ohr deprivation
group) had a median value of >60 min (C.I. 12->
60, n=36) similar to that of Fig. 4C. The CPPTs
for the 4 hr and 8 hr deprivation groups were >60
min (C.I. 40->60, n=27) and 17 min (C.I. 8-57,
n=31) respectively which were not significantly
different from that of the control partly because of
the large C.I.s. However, CPPT for 12 hr was 17
min (C.I. 4-43, n=27) which was significantly
shorter than the control. At 24 hr, CPPT reduced
markedly, as small as 4 min (C.I. 3-8, n=29). The
interval, however, did not further decrease in the
additional 48 hr of deprivation since CPPT was 4.5
min (C.J. 3-8, n=30) at 72 hr.
The shortening of CPPT may be due to a
reduction of spermatophore production as a result
To test this
possibility, males with accessory glands removed
were used. The accessory gland ablated males
could not make spermatophores but exhibit a
spermatophore protrusion-like behavior [1] some-
time after a vaccum copulation with the female [2—
5]. Thus, CPPT was defined here as the time
between genitalia separation and spermatophore
protrusion-like behavior. Fig. 8 shows the results
of the female-deprivation test in the “male” condi-
tion using accessory gland ablated males. CPPT of
the control (AG (0)) was 47 min (C.I. 10->60, n
of no copulation in the container.
964 T. OotsuBo AND M. SAKAI
Bas
oO
Median of CPPT (min)
ine)
oO
AG AG
(QO) (24)
Fic. 8. Effect of female-deprivation in males with
accessory glands removed on CPPT. AG(0), males
(n=30) with no deprivation as control. AG(24),
males (n=25) with 24 hr deprivation.
=30) while that of the 24 hr deprived group (AG
(24)) was 11 min (C.I. 7-25, n=25). It is obvious
that the shortening of CPPT occurred even in the
males lacking the ability of spermatophore produc-
tion. In addition, males whose testes were re-
moved showed a similar result with a CPPT of 7
min (C.I. 5-8, n=36). These results indicate that
the effect of female-deprivation can not be corre-
lated with the lowered activity of spermatophore
production.
The shortening of CPPT may be due to the lack
of genitalia coupling [1] and subsequent sperma-
tophore extrusion. Males were then placed with
females for 24hr whose subgenital plates were
closed with wax to prevent males from hanging the
epiphallus onto the female subgenital plate but not
to prevent other mating acts. They were restricted
in only genitalia coupling and spermatophore ex-
trusion. The result indicated that the shortening of
CPPT did not occur (CPPT>60 min (C.J. 28->
60, n=20)). This revealed that the shortening of
CPPT was not correlated with the inability of the
genitalia to couple and to extrude the sperma-
tophore.
It is worth mentioning that males reared with
males (they were not used so far in the experi-
ments) occasionally showed an extremely short
CPPT when they were tested in the “male and
female” condition. Although it was not apparent
in the males in Fig. 1C, in one case using a
different batch, CPPT was as short as 3 min (C.I.
3-4, n=28). The shortest of these was 1 min.
Such a short CPPT was seldom observed in males
reared with females (Fig. 1A). This reveals that
the effects of the female-chemical substances and
female-deprivation could be additive.
Effect of restraint in spermatophore protrusion
Table 2 shows the effects of the forced posturing
on spermatophore protrusion. Sixty-five per cent
of the “posture normal, leg contacted” males ex-
hibited spermatophore protrusion with a relatively
long latency (40 min (C.I. 19->60)), while only
10% of the “posture normal, leg restrained” males
did so. In contrast, no males of the “posture
reversed” groups ejected the spermatophores
within a | hr and the following 24 hr. These results
indicate that the male must be kept in a normal
posture to protrude the spermatophore with the
legs freely moving in contact with the substrate. In
an additional experiment, the male was im-
mediately paired with a female in a beaker at the
end of stress testing (“posture reversed, legs sus-
pended”). They showed spermatophore protru-
TABLE 2. Spermatophore protrusion under re-
strained conditions
Conditions Spermatophore :
Posture Legs protrusion (%) CPET. (ain)
Normal Contacted 65 [30] 40(C.1. 19->60)
Restrained 10 [35] 18, 55
Reversed Contacted 0 [0] —
Suspended 0 [0] —_
Number in each group, 20. Number in square
brackets, males produced spermatophore(s) within
24hr after the end of a 1 hr observation.
Spermatophore Protrusion in Crickets 965
sion with a CPPT of 9 min after pairing (C.I. 8-17,
n=20) which was not significantly different from
the control (8 min (C.1. 8-10, n=33)) tested simp-
ly under the “male and female” condition using the
same group. This indicates that spermatophore
protrusion was inhibited during heavy stress but
there was no aftereffect.
Transition from SGPO to SPPT
Disturbance
As shown in Fig. 2-C, the fact that the interval
between subgenital plate opening and sperma-
tophore protrusion was very constant suggested
that both actions were linked together. Thus,
whether spermatophore protrusion can be pre-
vented by heavily distracting the male immediately
after subgenital plate opening was examined. The
male was quickly picked up at subgenital plate
opening and kept gripped in the palm. In spite of
such a harsh treatment, all the males showed
spermatophore protrusion in the normal time in-
terval of 45 sec (C.I. 42-51, n=25).
Transection of the connnectives
To estimate the involvement of the anterior
nervous system in initiation and execution of sper-
matophore protrusion, the connectives were cut at
the neck by decapitation or at the junction be-
tween the thorax and the abdomen immediately
after subgenital plate opening. Both the groups
showed spermatophore protrusion with the normal
time interval of 45 sec (C.I. 41-48, n=20) and 45
sec (C.I. 42-51, n=20), respectively. These re-
sults revealed that spermatophore protrusion is an
unstoppable process once the subgenital plate
Opens and is accomplished under the control of
only the abdominal ganglia. In contrast, as will be
described below, when the connective transection
was made before subgenital plate opening, sper-
matophore protrusion within the normal time
range was prevented.
Effects of nerve and brain transection
When the male was kept with a female in a
beaker, spermatophore protrusion occurred reg-
ularly after every copulation. The number of
spermatophores reached an average of 16 per day
in males on day 10 after molt [5]. On the other
hand, when the male was kept alone in the beaker,
it showed spontaneous cycle renewal [1] in which
the male exhibited spermatophore protrusion
without any mating actions. In the present experi-
ments, 96% of the control males (Fig. 9, a) pro-
duced one or two spermatphores per day with an
average of 1.6+0.7. No males (Fig. 9, b) with the
bilateral connectives cut between the 4th and the
Sth abdominal ganglion produced sperma-
tophores. Only males in this group showed feces
discharge all the time indicating that they had
problems in the control of intestinal and abdomin-
al movements. In contrast, males with a unilateral
connective cut at the same level produced sperma-
tophores similarly to the control. In males (Fig. 9,
c) with bilateral connectives cut between the tho-
rax and abdomen, the average number of sperma-
tophores was about half the control. Unilateral
transection again altered neither the number of the
males producing spermatophores nor the number
of spermatophores produced.
Decapitated males (Fig. 9, d) and decerebrated
males (Fig. 9, e) also showed a significant decrease
in the numbers of males producing sperma-
tophores (71% and 50%) and the number of
spermatophores produced (1.0+1.4 and 0.4+0.5)
compared with the control.
The brain was cut in different ways to separate
the particular regions. Horizontal transection
(Fig. 9, f) caused a significant decrease in the
numbers of males producing spermatophores
(65%) and spermatophores produced (0.6+0.8).
Males with the antero-median region eliminated
(Fig. 9, g) had a significant decrease in both the
numbers of males (68%) and spermatophores (0.5
+(0.6). On the other hand, males with the antero-
lateral regions eliminated (Fig. 9, h) showed only a
significant derease in the number of sperma-
tophores (0.8 +0.6) with a similar number of males
producing spermatophores (79%) as the control.
The median transection (Fig. 9, j; n=22) caused a
significant decrease in the number of sperma-
tophores produced but not in the number of males
producing them (88%). Finally, the removal of the
corpora allata and the corps cardiacum (Fig. 9, i; n
=12) did not alter the number of males (91%) or
spermatophores (1.3+0.8).
Only the disconnection of the bilateral nerve
966 T. OotsuBo AND M. SAKAI
Males with spermatophores (%)
Mean of spermatophores / day
O
b © a e f
el, MY,
wv
oO
neon 5 A
pecs case
Fic. 9. Effects of transection of the connectives, separation of the brain, and removal of the corpora allata and
corpus cardiacum. A: percentage of males producing mature spermatophores at least once in 3 successive days
following surgery. B: the average number of mature spermatophores produced per male per day. Small letters
(b-j) represent the types of surgery corresponding to those in inset. Each group consisted of the following number
of males; 24(a), 13(b), 20(c), 14(d), 12(e), 20(f), 22(g), 19(h), 12(i), and 22(j). Asterisks indicate significant
differences (A: 2? test, P<0.05; B: t-test, P<0.05) between (a=control) and (b-j).
cords between the 4th and the 5th abdominal
ganglion abolished spermatophore protrusion.
These results indicate that spermatophore protru-
sion can be initiated and executed without any
influence from the head or throacic ganglia.
DISCUSSION
The present study shows the starting event of
spermatophore protrusion, and external and inter-
nal factors that influence the latency of sperma-
tophore protrusion in the male cricket Gry/lus
Spermatophore Protrusion in Crickets 967
bimaculatus. Initiation of spermatophore protru-
sion is considerably facilitated both by contact with
the female and by an increase in sensitivity to the
female. On the other hand, spermatophore
protrusion was delayed or prevented under stress-
ful conditions such as persistent restraint in an
abnormal posture. Spermatophore protrusion
could occur, though more slowly, in males with the
connectives cut, except connectives between the
4th and the 5th abdominal ganglion. Our results
provide the basis for future neurophysiological
study to elucidate the initiation mechanism of
spermatophore protrusion.
Start of spermatophore production
Spermatophore protrusion occurs with a more
variable CPPT compared with that (about 1 hr) of
the calling song [1]. In extreme cases, CPPT was
more than 1 hr (see Fig. 2B) while only 1 min at
the shortest. These findings suggest that the pro-
cess of spermatophore protrusion is influenced by
some external and internal factors in contrast to
the process of the calling song which is dependent
on an internal timer [4].
Spermatophore protrusion occurs 45 sec after
subgenital plate opening. Transection of the con-
nectives revealed that these two actions were a
continuous process. Inspection of the sperma-
tophore in the ejaculatory duct indicated that the
secretion of sperm from the testes and proteinous
substances from the accessory glands occurred at
the moment of subgenital plate opening. These
facts indicate that spermatophore protrusion,
which we defined a priori as the moment of
immature spermatophore ejection through the
genital cavity to the exterior, actually starts at the
moment of accessory gland contraction to secrete
the spermatophore materials into the ejaculatory
duct [14] possibly mediated by dorsal unpaired
median (DUM) neurons [15]: i.e. the male cycle
starts at the time of subgenital plate opening.
Previously, one paper [4] reported that a new
spermatophore was already present in the ejacula-
tory duct soon after spermatophore protrusion and
stayed there until the next spermatophore protru-
sion, while the other paper [14] did not confirm
this and rather agreed with our present result. We
suspect the observations by the former authors
might be an artifact due to freezing and thawing.
Facilitation of spermatophore protrusion
CPPT was much shorter in the male kept con-
tinuously with the female after copulation. This
suggests that some contact-mediated chemical sub-
stances are facilitating spermatophore protrusion.
Chemo-receptors mediating spermatophore
protrusion may be distributed over the body sur-
face because the shortening of CPPT was still
apparent in males without the antennae, palpi and
tarsi. Previously in Teleogryllus, the female had no
effect on spermatophore protrusion [3] though its
contact chemoreceptors certainly facilitated mat-
ing actions [16]. The discrepancy may be due to
the difference in species or the difference in the
size of the space where animals were tested. The
compartment they used was a little wider than our
200 ml beaker, which might have lowered chances
of contact with each other. It is interesting that
males split into those with CPPT less than 30 min
and those with more than 1 hr when tested in no
female conditions (Fig. 4B, C). Probably an indi-
vidual difference in male reproductive activity was
exaggerated through some accommodation which
increased the threshold of spermatophore protru-
sion under less stimulative conditions.
Spermatophore protrusion was also facilitated
after males were temporarily kept isolated from
females. This was already seen from day 3 after
molt, and matched our empirical observations that
males reared without females were sexually more
excitable than those with females [1]. Our sys-
tematic study of female-deprivation found that the
shortening of CPPT became pronounced after a
12-24 hr separation. Our results indicated that
this effect was neither due to a reduction of
spermatophore production nor due to the lack of
genital coupling and spermatophore extrusion dur-
ing isolation. It is probably caused by an increase
in sensitivity to the chemical substances of the
female.
Inhibition of spermatophore protrusion
Stress, such as forced posturing, significantly
delayed or even prevented spermatophore protru-
sion. In particular, males fixed in the upside-down
position were unable to push out the sperma-
968 T. OotsuBso AND M. Sakal
tophores, indicating that normal posture with the
legs kept freely on the substrate is a prerequisite.
A similar result was previously obtained by fixing
the body to a board with paraffin [4]. These results
give cautions against neurophysiological study
where animal restraint would be inevitable. Simi-
lar but not large delay in CPPT was also found
under a different type of stress, 1.e., fighting with a
male (Fig. 4B).
It should be worth comparing the resistivity to
stress between the two interval states: genitalia
separation to spermatophore protrusion vs. sper-
matophore protrusion to calling song. As long as
the male is under heavy stress, it is natural that the
male cannot initiate either spermatophore protru-
sion or calling song. However, the male sang the
calling song with the normal latency (about 1 hr)
from spermatophore protrusion when stress was
terminated in less than 1 hr after spermatophore
protrusion [4]. Stress did not disturb the running
timer. On the other hand, the male exhibited
spermatophore protrusion with a latency of 1h
plus the normal CPPT when stress was terminated
1 hr after copulation and the male was paired with
a female. Stress inhibited the processing necessary
for spermatophore protrusion.
Role of the brain in spermatophore protrusion
It is known that nervous communication be-
tween the brain and the terminal abdominal gan-
glion is necessary for the male cricket to perform
mating behavior normally [9]. Here, we state
more concretely that male crickets with both con-
nectives cut at the thoracic level can perform
spermatophore protrusion but not produce as
many spermatophores as intact males. This result
means that the motor program for spermatophore
production resides totally within the abdominal
ganglia and can proceed without command of the
anterior ganglia. Normally, however, sperma-
tophore protrusion is certainly triggered by a com-
mand in the brain because decerebrated males
failed in spermatophore protrusion within the nor-
mal time range after copulation.
It would be interesting to record the descending
neural activity conveying signals for sperma-
tophore protrusion which should arrive at the
abdominal ganglia before subgenital plate open-
ing. The fact that spermatophore protrusion re-
quired 45 sec from subgenital plate opening and
that the minimum CPPT (genitalia separation to
spermatophore protrusion) was only | min suggest
that information on mating termination generated
in association with the dorsal pouch contraction to
extrude the spermatophore [1] can be processed
immediately in the brain and in turn can trigger the
pattern generator in the abdominal ganglia. At the
same time, some neural events associated with
DUM neuron activity for secretion of the sperma-
tophore materials into the ejaculatory duct may set
the timer for the calling song. The resultant
accessory gland contraction itself or the ensuing
spermatophore ejection to the external genitalia is
not concerned with the timer setting since males
without accessory glands [2] and those with sper-
matophores but incapable of spermatophore
protrusion [1] still retain the normal timer func-
tion.
Regarding the effects of partial elimination of
the brain, determination of brain regions critical
for spermatophore protrusion was virtually im-
possible because the activity of spermatophore
production in intact males was very low (1 or 2 per
day) and that in the experimentals was not zero but
0-1 per day: the differences were too small to
compare. The total inability of males with the
connectives transected between the 4th and the Sth
abdominal ganglion to perform spermatophore
protrusion is probably due to the paralysis of
abdominal segments as suggested by continuous
discharge of feces. The results of the corpora
allata and corpus cardiacum removal was in agree-
ment with the previous report that elimination of
either organ failed to abolish mating behavior [2].
ACKNOWLEDGMENTS
The study was supported by Grant-in-Aid (No.
2640557) for Scientific Research from the Japanese
Ministry of Education, Science and Culture.
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ZOOLOGICAL SCIENCE 9: 971-981 (1992)
© 1992 Zoological Society of Japan
Ultrastructure of Spermatozoa and Spermatogenesis in Nepomorpha
(Insecta: Heteroptera) with Special Reference to Phylogeny
YouNG Hwan Lee! and CHANG Eon LEE*
'Department of Biology Education, College of Education, Taegu University,
Kyungsan 713-714, and *Department of Biology, College of Natural
Sciences, Kyungpook National University, Taegu 702-701, Korea
ABSTRACT — Ultrastructural study of spermatozoa was made for five species and four genera in two
families of Nepomorpha, Belostomatidae and Nepidae (Insecta: Heteroptera).
The following five
synapomorphic relationships were recognized for the fine structures of spermatozoa: 1) Nepomorpha
have acrosome displaced to a lateral position with respect to the nucleus; 2) the centriole adjunct is well
developed in mature spermatozoa; 3) each of the two mitochondrial derivatives contains two or three
crystalline bodies; 4) the derivatives are linked to the axoneme by two bridges at the level of doublets 1
and 5; and 5) accessory bodies are absent in this group. A brief comments on the phylogenetic
relationships among three families (Belostomatidae, Nepidae and Notonectidae) of Nepomorpha is
given.
INTRODUCTION
The aquatic heteropteran group Nepomorpha
(=Cryptocerata or MHydrocorisa; Insecta) re-
corded in Korea comprise 32 species, belonging to
16 genera of 8 families. Lee [1] attempted the
reconstruction of Nepomorpha phylogeny based
on adult characters, in particular fine structures of
genitalia.
Though comparative spermatology seems to
provide useful information for heteropteran sys-
tematics, only few papers have been published on
Nepomorpha. Itaya er al. [2] described fine struc-
tures of the later stages of spermiogenesis in
Leptocoris trivittatus Provancher (Corixidae), but
did not refer to mature spermatozoon. Descrip-
tions of the sperm in Nepa rubra L. (Nepidae)
were given by Werner [3].
In Nepomorpha as a whole, a few works on
mature spermatozoa of the following species have
been made: Diplonychus esakii Miyamoto et Lee
[4] (Belostomatidae), Ranatra unicolor Scott [5]
and Laccotrephes japonensis Scott [6] (Nepidae)
and Notonecta glauca L. [3, 7-9] (Notonectidae).
In Notonectidae no information on the centriole
Accepted May 30, 1992
Received October 26, 1991
adjunct is available. Recently Lee and Lee [5]
reported the centriole adjunct of mature spermato-
zoa of Ranatra unicolor Scott.
In this paper we report and compare some
ultrastructures of the spermatozoa and sperma-
togenesis in Nepomorpha.
MATERIALS AND METHODS
The following five species of Nepomorpha were
examined in this study: Belostomatidae—
Muljarus japonicus, Diplonychus esakii; Nepidae
—Ranatra unicolor, R. chinensis, Laccotrephes
Japonensis. They were all collected near Taegu,
Korea.
For transmission electron microscopy, testes and
seminal vesicles of the adult males were fixed in
2.5% glutaraldehyde in a 0.1 M cacodylate buffer,
rinsed in 0.1M cacodylate buffer, pH 7.2, post-
fixed in 1% osmium tetroxide for 40 minutes,
dehydrated in ethanol, and embedded in Epon
812. Ultrathin sections were stained with uranyl
acetate and lead citrate and examined in a JEOL
100 S and a Hitachi H-600.
For light microscopy, a drop of suspension of
spermatozoa obtained by squeezing the seminal
vesicle in cacodylate buffer was smeared on a slide,
covered with a slide cover and photographed with
972
Olympus multipurpose microscope.
RESULTS AND DISCUSSIONS
Morphology of the spermatozoa and sperma-
togenesis in Nepomorpha
Spermatozoa and the structural changes that
Y. H. LEE anp C. E. LEE
occur during spermatogenesis In the five species of
Nepomorpha have been examined. The spermato-
zoa of the Nepomorpha vary in length in different
species, from 210 4m or less to 16.5 mm or more.
The head is from 20 4m to 1.16 mm long (Table 1).
1.1 Belostomatidae
Muljarus japonicus has spermatozoa measuring
B: Belostomatidae; Ne: Nepidae; N: Notonectidae.
TABLE 1. Summary of the morphology of the sperms and spermatogenic cells in 6 species of Nepomorpha
B Ne N
M D R R L N Character state Character
j e u c j g
= = 200-220 um or less =
— 221-240 um
= 241-260 ~m '— Total length of the sperm
= 350-370 vm
— 15mm or more H
=z = | 30-40 ~m or less —
_ SL SU att ‘— Head length
= 61-70 em
= 1mm or more =
oe ee eee ee |e Lateral disposition o-
—_— | 0.1-10 pm? ~— '— Acrosome
— | 11-50 zm?
= 51-100 zm? i— volume +
a | 10,000 pm
or more —
= oe eee | eee Compact _—
Texture of acrosome
a Not compact =
Absent a
ett Cane ce Microtubules within acrosome
Present aan
mee eee eee ee oe Tubular type Subacrosomal lumen
oe ee fee ees |S Circular type — :
; Nuclear section
— | Kidney type a
om ee eee ee |CND Long = ’
Centriole adjunct
=e ee Abundant =
|
oe eee eee eee see | Axoneme (9+ 9+2) aa
em ee oe Bridges between A and MD -— Tail
oe eee eee ee ee MD crystallization ~
Equal —
Eh a ee i MD symmetry
wee | Not equal =
(2 ee ee ee ||!
Mj: Muljarus japonicus; De: Diplonychus esakti; Ru:
Ranatra unicolor; Re: Ranatra chinensis, Lj: Loccotrephes japonensis, Ng: Notonecta glauca, ND: no data.
Spermatogenesis of Nepomorpha 973
about 260 “m in total length (Fig. 1), 0.4 ~m in
diameter at the head and 0.6 um at the tail, the
head being 30-35 um long. D. esakii has sperma-
tozoa measuring 210 “m in total length, the head
being 65 um long (Fig. 10). Spermatozoa of D.
esakii are longer and the heads are shorter than
those of M. japonicus.
The acrosome is displaced to a lateral position
with respect to the nucleus in the family (Fig. 2) as
well as in the other examined groups of Nepomor-
pha (Figs. 11, 16, 26, 30). The family has an
acrosome of very small size, approximately 1.4
yam®* in volume in the early spermatid of Muljarus
japonicus (Fig. 6) and 0.9 um in Diplonychus
esakiit. The acrosome of M. japonicus is composed
of an electron-dense material during the spermatid
differentiation (Fig. 6), whereas that of D. esakii is
very low in electron density (Fig. 11).
The nucleus of the sperm shows kidney-shape in
its cross section during the differentiation of the
spermatid (Figs. 3, 13). The morphology of the
nucleus is distinctly different when compared with
those of other families of Nepomorpha (Table 1).
The nuclear membrane holds in its posterior con-
cavity the centriole adjunct in early spermatid and
they form the nucleus-centriole adjunct complex
(Fig. 5).
The spermatid tail elongates tangentially at the
proximal portion of the nucleus (Fig. 4). The
centriole adjunct is abundant and displaced to a
lateral nucleus in mature spermatozoa (Figs. 8,
12). The sperm of this family has a “9+9-+2”
axonemal pattern (Figs. 9, 14). A sperm bundle
contains 1024 spermatozoa (Fig. 7), the mitosis
occurring eight times at the spermatogonia.
1.2 Nepidae
Ranatra unicolor spematozoa are about 225 um
long (Fig. 15); the head about 20 ~m long and 0.5
ym wide at the base and 0.4 wm wide at the tip.
The mature acrosome is approximately 5.83 ~m
long and 0.33 um thick (Fig. 16). The acrosome
contains 220-250 A wide longitudinal tubules
through its length (Fig. 17) [5]. The acrosome of
the early spermatid is approximately 30 ~m* in
volume (Fig. 18).
R. chinensis spermatozoa are about 220 4m long
(Fig. 24); the head about 20 um _long and 0.8 ~m
wide at the base, 0.5 ~m wide at the tip. The early
acrosome of approximately 20 7m? in volume is
smaller than that of R. unicolor.
The filiform spermatozoa of
japonensis are about 370 um long (Fig. 29); the
head about 45 ~m long and 0.33 ~m wide at the tip
and 0.5 ~m wide at the base. A sperm bundle of
Laccotrephes
L. japonensis contains 512 spermatozoa (Fig. 35).
The acrosome forms a spear with the hooked tip.
The posterior surface of the acrosome invaginates
to the one side of the nucleus (Fig. 30). The
acrosome of the early spermatid is approximately
2.5 wm in diameter and 65.4 ~m* in volume (Fig.
36), which is the largest one of the Nepidae.
The acrosome of Nepidae is much longer and
larger than that of Belostomatidae and also forms
an apical cap, extending on one side along the
anterior nucleus (Figs. 16, 30). The family has a
prominent acrosome which contains tightly packed
tubules (Fig. 17). An acrosome with tubules has
been described from Gerromorpha but not from
acrosomes of other animals [10, 11]. Dallai and
Afzelius [12] have proposed that this type of
acrosome represents an adaptation to life in fresh
water. The subacrosomal space forms an elon-
gated tube extending to its anterior end through
the acrosome.
The nucleus is a cylindrical rod with highly
condensed chromatin, but apically is narrowed and
surrounds the acrosome which is in cross section
embedded in an opened thick ring of the nucleus
(Figs. 19, 26, 31). The anterior and posterior
nuclear cross-sections contain the acrosome and
the centriole adjunct of crescent-shape respective-
ly (Figs. 21, 26, 27, 34).
The family has well-developed centriole adjunct
of very elongated and electron dense structure in
mature sperm (Figs. 20, 21, 25, 27, 33, 34). The
centriole adjunct of R. unicolor lengthens to at
least 10 ~m more in nearly mature spermatozoa
(Fig. 20). It extends anteriorly lateral to the basal
nucleus and surrounds posteriorly the outside of
the mitochondrial derivatives and flagellum.
The lateral position of the centriole adjunct is
very characteristic in Nepidae as well as Belosto-
matidae. In particular two families have abundant
centriole adjunct displaced to a lateral nucleus in
mature spermatozoa (Figs. 5, 8, 12, 20, 25, 33).
Y. H. LEE AND C. E. LEE
974
Spermatogenesis of Nepomorpha 975
Fics. 1-9. Muljarus japonicus (Belostomatidae).
Abbreviations in these and subsequent figures: A=acrosome; Ax=axoneme; N=nucleus; CA=centriole adjunct;
MD=nmitochondrial derivatives
x 30,000.
Fic. 1. Mature spermatozoa. Scale bar; 25 um.
Fic. 2. Acrosome displaced to a lateral nucleus. 6,000.
Fic. 3. Cross section of nucleus in spermatid. 18,000.
Fic. 4. Tail elongating tangentially at the proximal portion of the nucleus.
Fic. 5. Longitudinal section of head-tail junction. x 15,000.
Fic. 6. Acrosome of the early spermatid. x 10,000.
Fic. 7. Cross section of a sperm bundle. 2,300.
Fic. 8. Cross section of nucleus-centriole adjunct complex. 20,000.
Fic. 9. Cross section of tail. 30,000.
The nucleus-centriole adjunct complex follows two
mitochondrial derivatives which surround the
axoneme. The microtubules are ordered in hex-
agonal and clover arrangement around the nuc-
leus-centriole adjunct complex during the sperma-
tid differentiation (Figs. 23, 37).
The tail appears symmetrical. It is made up of
an ordinary axoneme and two mitochondrial de-
Tivatives of equal size. These components are
arranged throughout the length of the tail. The
mitochondrial derivatives are the bilateral sym-
metry of the axoneme (Fig. 22). They surround
most of the axoneme in Nepidae (Figs. 22, 28, 32).
The horizontally oriented cristae exhibit a very
regular periodicity of approximately 55—-S7 nm in
R. unicolor (Fig. 20), 47 nm in R. chinensis (Fig.
25) and 41 nm in Laccotrephes (Fig. 33).
The axoneme belongs to the basic 9+-9+-2 pat-
tern, having evident radial links with prominent
heads, evident dynein arms on the doublets, and a
976 Y. H. Lee AND C. E. LEE
a
Be
Fics. 10-14. Diplonychus esakii (Belostomatidae).
Acrosome showing very low electron density in spermatid. 42,000.
x 69,000.
Fic. 10. Mature spermatozoa. Scale bar; 25 um.
Fic. 11.
Fic. 12. Cross section of nucleus-centriole adjunct complex.
Fic. 13. Cross section of nucleus in spermatid. X 12,000.
Fic. 14. Cross section of tail. 49,000.
layer of 9 accessory tubules. The diameter of the
crystallization of the mitochondrial derivatives
may measures more or less half of the section in
the all examined group (Figs. 9, 22, 28, 32). The
axoneme is flanked by two mitochondrial deriva-
tives but lacks accessory bodies, such as are found
in spermatozoa from Homoptera and many related
groups of insects [13].
Three further features are also characteristic in
Nepomorpha as follows: (1) the presence of two
bridges that join the mitochondrial derivatives to
the axoneme at the level of the microtubular
doublets nos. | and 5; (2) the occurrence of two or
three crystalline bodies in the mitochondrial de-
rivatives, rather than a single one as is common in
pterygotes; (3) the abundant centriole adjunct in
mature sperm. The third feature seems to be
unique to spermatozoa of Nepomorpha, such as is
found in spermatid of Pentatomidae but has not
been observed in mature sperm [14].
1.3 Notonectidae 7
Estimations of the sperm length have ranged
between 14mm for Notonecta maculata [15] to
16.5 mm for N. glauca [16]. They are the longest
one among the animal kindgom. N. glauca de-
scribed previously [3, 7-9] are reanalysed in the
light of phylogeny of Nepomorpha.
Two special structures appear in connection with
the nucleus during spermatogenesis in Notonecta
species. These are pseudochromosomes in sper-
matocytes and nuclear calottes in mid spermatids.
Spermatogenesis of Nepomorpha
Fics. 15-23. Ranatra unicolor (Nepidae).
Fic.
Fic.
Fic.
Fic.
Fic.
Fic.
Fic.
Fic.
Fic.
1),
16.
7.
18.
19.
20.
21.
22.
23.
Mature spermatozoon. Scale bar; 50 ~m.
Longitudinal section of acrosome. X 12,000.
Cross section of acrosome containing tightly packed tubules. 30,000.
Acrosome of the early spermatid. 12,000.
Cross section of the head. 24,000.
Longitudinal section of centriole adjunct. 8,000.
Cross section of nucleus-centriole adjunct complex. 24,000.
Cross section of-tail. 60,000.
Cross section of microtubules between the nucleus and a membranous sleeve.
x 30,000.
Y. H. Lee ann C. E. LEE
Longitudinal section of sperms showing nucleus-centriole adjunct complex and tail. 30,000.
Fics. 24-28. Ranatra chinensis (Nepidae).
Fic. 24. Mature spermatozoon. Scale bar; 25 um.
Fic. 25.
Fic. 26. Cross section of head. 32,000.
Fic. 27. Cross section of nucleus-centriole adjunct complex. 32,000.
Fic. 28. Cross section of tail. 32,000.
In spermatocytes the membrane system consists of
a shell of two or three parallel cisterns enclosing
the whole nucleus at a certain distance from its
envelope. In young spermatids remnants of the
former shell change to spherical calottes and be-
come attached to the nucleus. After separation
from the nucleus it ultimately disintegrates to
whirls of cisterns still visible in late spermatids [8].
The acrosome grows to a sphere measuring
about 20 um in diameter and 33493.33 ~m®* in
volume. During elongation the acrosome attains
spindle shape, the lateral process gradually becom-
ing its anterior part [7]. The acrosome of the
backswimmer resembles somewhat that of two
other hemipterans, Nepa rubra L. [17] and Gerris
remigis [10]. At its posterior end the acrosome has
a lateral groove. It appears empty at some levels
but contains the nucleus and the centriole at a
more posterior level.
The axoneme is of a common type among insects
with a 9+9-+ 2 pattern, and it is unusual only by its
great length. The larger mitochondrion occupies
Spermatogenesis of Nepomorpha
Fics. 29-34. Laccotrephes japonensis (Nepidae).
Fic. 29.
Fic. 30.
Fic. 31.
Fic. 32.
Fic. 33.
Fic. 34.
Mature spermatozoon. Scale bar; 50 um.
Longitudinal section of head. 16,000.
Cross section of head. 9,000.
Cross section of tail. 30,000.
979
Longitudinal section of sperms showing nucleus-centriole adjunct complex and tail. 12,000.
Cross section of nucleus-centriole adjunct complex.
x 30,000.
980 Y. H. Lee
AND C. E. LEE
Fic. 35. Cross section of a sperm bundle. 4,000.
Fic. 36. Acrosome of the early spermatid. 10,000.
Fic. 37.
x 18,000.
70-80% of the tail cross-section, the smaller one
10-15%. The two mitochondria are completely
embracing the axoneme. The mitochondria have
regularly arranged cristae. The three paracrystal-
line bodies can be recognized in both the small and
the large mitochondrion. The shapes of the bodies
differ in the two mitochondria. The paracrystalline
bodies occupy most of the mitochondrial volume
[7].
2. Phylogenetic relationships among three fami-
lies in Nepomorpha based on the spermatozoal
ultrastructures
In an investigation of spermatozoal ultrastruc-
ture from six species of Nepomorpha, the position
of the acrosome and centriole adjunct appears to
be specific for Nepomorpha and the centriole
adjunct is well developed in mature spermatozoa.
Diplonychus esakti and Muljarus japonicus are
Cross section of microtubules between the nucleus-contriole adjunct complex and a membranous sleeve.
easily distinguished by the following characteris-
tics: (1) the total length of the sperm of M.
Japonicus is longer than that of D. esakii, on the
other hand the head length is shorter than that in
D. esakii; and (2) the acrosome has low electron
density in the spermatid of D. esakii, while M.
japonicus has a dense electron acrosome through
the sperm differentiation. The differences be-
tween the two species are evident and they are not
closely related, although they are grouped to the
same genus, until Lee [1] established a new genus
Muljarus for the species of japonicus groups.
Two species of Ranatra are closely related by the
characteristics showed in Table 1. Laccotrephes
has many resemblances to Ranatra in nucleus,
acrosome and centriole adjunct and two genera
should be related to each other closely grouped.
Both Belostomatidae and Nepidae are closely
related based on the ultrastructures of acrosome,
Spermatogenesis of Nepomorpha 981
subacrosomal lumen, centriole adjunct and mito-
chondrial derivatives, though they are different in
the morphology of the nucleus during the dif-
ferentiation and in the microtubules within the
acrosome. On the contrary, Notonecta are char-
acterized by the ultrastructure of mitochondrial
derivatives with two different sizes and the giant
sperm and the acrosome.
Miyamoto [18] considered that the Nepidae and
Belostomatidae are of the same evolutional direc-
tions in the structures of the alimentary organs.
Cobben [19] lined Nepidae with Belostomatidae
based on the distribution of the larval abdominal
glands and the structures of stylets. Lee [1] also
grouped the Belostomatidae with Nepidae based
on the genital structures and subdivided Nepomor-
pha into four family groups, Belostomatidae and
Nepidae; Notonectidae, Pleidae and Helotrepi-
dae; Aphelocheiridae, Naucoridae, Gelastocor-
idae and Ochteridae; and Corixidae. Moreover he
suggested that they do not show any phylogenic
trend in genital structures of the four groups and
the different characters might have been derived
from the polyphyletic terrestrial origins.
ACKNOWLEDGMENTS
This study was supported in part by the Basic Science
Research Institute Program, Ministry of Education,
Korea, 1991.
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3 Werner, G. (1986) Change in the Golgi apparatus
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ZOOLOGICAL SCIENCE 9: 983-988 (1992)
© 1992 Zoological Society of Japan
Self-Nonself Recognition in the Colonial Protochordate
Botryllus schlosseri from Mutsu Bay, Japan‘
Barucu RINKEVICH~ and YASUNORI SAITO”
National Institute of Oceanography, Israel Oceanographic & Limnological
Research, Tel-Shikmona, P.O.B. 8030, Haifa 31080, Israel and
3Shimoda Marine Research Center, University of Tsukuba
5-10-1 Shimoda-shi, Shizuoka 415, Japan
ABSTRACT— Wild Botryllus schlosseri collected from a 5X5 m area in Mutsu Bay (Aomori Prefecture,
Japan) were tested for alloresponses in intrapopulation colony allorecognition assays (CAAs). Results
indicate that rejection patterns are similar to those recorded previously in the populations from
Monterey and Santa Barbara, California, from the Mediterranean coast of Israel, and from the
Venetian lagoon, Italy. The only difference was the marked accumulation of bright-yellow blood cells in
the tips of interacting ampullae. Pairwise CAAs which were performed on all combinations (n=91)
from 14 colonies resulted in 12.1% fusions, which gives a populational estimation of 32 alleles on the
fusibility locus. A fusibility chart for these 14 genotypes revealed 18-19 different, not equally frequent,
allorecognition alleles, of which 12 were assigned to only one genotype, each; 5 occurred twice, and each
one of 2 alleles was present in three different genotypes. It is concluded that the sampled area was too
small to represent the probable higher number of fusibility alleles residing in this population.
INTRODUCTION
It is now almost 90 years that studies on the
colonial tunicate Botryllus schlosseri (Pallas) have
shown its capacity for colony specificity [1], a
histocompatibility system which resembles the ma-
jor histocompatibility complex (MHC) of the
vertebrates in many aspects [2]. The genetic basis
for the colony specificity resides in a single, highly
polymorphic haplotype (called the fusibility /histo-
compatibility locus, Fu/HC [3]), which possesses
multiple codominantly expressed alleles. Interact-
ing colonies which do not share any allele on this
locus reject each other, while colonies which share
in common at least one allele on the Fu/HC locus
may undergo a natural transplantation (fusion) by
forming vascular anastemoses between their
peripheral ampullae [2, 4-6].
B. schlosseri is a cosmopolitan inhabitant of
shallow water, hard bottom communities [7, 8].
Accepted July 1, 1992
Received April 16, 1992
' Contribution number 541 from the Shimoda Marine
Research Center.
* To whom all correspondence should be addressed.
This species has been studied for intrapopulation
allorecognition responses in 4 remote localities:
the population from the Venetian lagoon, Italy [6,
9], from the Mediterranean coast of Israel [10],
from Woods Hole, Massachusetts, Atlantic Ocean
[2, 11, 12], and from the Monterey and Santa
Barbara areas, California, Pacific Ocean [11-15].
Results indicated that allogenic interactions be-
tween ampullae of noncompatible Botryllus con-
specifics exhibited not only the species-specific
characteritics of rejection processes, but also
population-specific rejection types which were
constantly expressed even in interpopulation en-
counters [11, 12]. When two incompatible colonies
of the Woods Hole population came into tunic-
tunic contact, a limited fusion of the cortical layers
of both colonies prevailed, which resulted in a
continuous tunic matrix between both partners.
This permitted a reciprocal ampullae penetration
which was followed by ampullae amputation and/
or haemorrhages formation, the development of
dark-brown necrotic areas, points of rejection
(PORs). In the other 3 studied B. schlosseri
populations, on the other hand, the outer layers of
the tunics did not fuse during allogeneic encoun-
984 B. RINKEVICH AND Y. Saito
ters. As a result, the cortical layers were clearly
observed as demarcating lines between colonies.
PORs, therefore, were developed without recip-
rocal ampullae penetration.
The above studies [12, 13] have suggested that
an extended comparison of intrapopulation
allogeneic interactions would be of great benefit
for a better understanding of self/nonself histo-
compatibility alloresponses of this cosmopolitan
species. Here we study allorecognition of a B.
schlosseri population from Mutsu Bay, Japan, and
try to evaluate the polymorphism pattern of the
Fu/HC locus by calculating frequencies of fusion
within a population sampled from a small area.
MATERIALS AND METHODS
Wild B. schlosseri colonies were collected
(November 1991) in Mutsu Bay, Aomori (Aomori
Prefecture), where they were grown in shallow
water on floating Pecten culturing cages. All
colonies were collected from a5 x5 m area. Large
healthy colonies were removed from substrates by
razor blades, tied with thin cotton fibers onto 2.5 x
7.5cm glass slides, and shipped to Shimoda
Marine Research Center, where they were kept
vertically in slots within a wooden culture box,
submerged in Nabeta Bay, Shimoda. For the
colony allorecognition assays (CAAs), we used
small groups of zooids at the growing edges of
colonies, carefully isolated from colonies. These
subclones were attached in pairs on 5X7.5 cm
glass slides, as described previously [11-15]. Dur-
ing the experiments, the colonies were kept in
17-liter standing seawater tanks, aerated by air-
stones and maintained at 17-18°C by aquarium
heaters. CAAs were observed daily and cleaned
during the observations by soft small brush. Food
was supplied daily (artificial diet; Liquifry Marine,
England).
RESULTS
Intrapopulation alloresponses
Contacts between extended ampullae of encoun-
ter colonies were established within 24 hr after
forming the CAA. Tips of marginal ampullae were
reciprocally positioned in tip-tip orientation where
the cortical layers of the allogeneic partners were
clearly observed as demarcating lines along contact
areas. When compatible pairs were assayed, the
tunic matrices eventually fused in limited or in
broader areas. Ampullae of both partners, or of
only one partner in a pair, penetrated into the
tunic matrix of the other colony through the fusion
areas and were positioned in tip-ampulla base
(proximal part) orientations. This resulted, within
less than 24 hr, in allogeneic anastomosis of blood
vessels, the formation of chimeras. Chimeras were
followed up for a period of one month. During this
period, chimerism in several assays already ended
in the resorption of all zooids of one partner each.
When nonfusible partners come into direct con-
tact, tunics did not fuse together, so ampullae
engaged each other reciprocally but indirectly,
through both cortical layers (Fig. la-i). During
this process, the tips of interacting ampullae, and
sometimes also the tips of all peripheral ampullae
(even those of the other sides of the colony), of
one or both partners in a pair, became very
distinctive in bright fluorescent yellow color. This
resulted from aggregations of bright yellow blood
cells which accumulated in the ampullar tips (Fig.
la, b). This phenomenon was also observed in
interacting colonies of the Monterey population
[11, 12] and the Mediterranean colonies [10]. By
employing histological examinations, we (Terta-
kover and Rinkevich, unpublished) characterized
the accumulated cells as morula cells. However,
the Mediterranean and Monterey populations did
not exhibit the deeper intensity in color and the
high frequency of cases as it is recorded in the
Japanese population. In most of the yellow col-
ored tips, the intense yellow color gradually abated
and disappeared within the next 24—72 hr, while in
some of the tips the color became dark-brown,
blood cells infiltrated out of the ampullar tips, and
the formation of PORs through haemorrhages was
documented (Fig. la-i). In a few cases, ampullae
were amputated from the peripheral blood vessels
and gradually disintegrated. Only a few (1-8) out
of many (up to tens) of interacting ampullae
produced cytotoxic lesions (Fig. la, e). Following
the acute phase of allogeneic response, where all
PORs were developed within a short period of a
Allorecognition in Japanese Botryllus 985
Fic. 1. Allogeneic interactions between B. schlosseri colonies: genotype combination 3 (left) vs 2 (a-d), and
genotype combination 10 (left) vs 3 (e-1). a-48 hr after CAA. Six small reciprocal PORs along the contact area
(arrowheads) out of 12 vs 17 interacting ampullae. Tips of ampullae (genotype 3) still possessed aggregations of
bright yellow cells in the contact area and in other peripheral ampullae (confined between two white arrowheads).
_b-Two days thereafter. No more PORs were added. Ampullae were cleared of yellow cell aggregations, and only
9 vs 7 were still interacting. Genotype 2 started retreat growing [14]. c-Two days later. Only a few colony 2
ampullae were still in contact with those of colony 3. Colony 2 zooids retreated 1-2 mm. d-7 days later. Zooids
of colony 2 retreated more than 3 mm from the initial location, leaving an unvascularized tunic matrix with
rudiments of blood vessels/ampullae and masses of degenerated infiltrating blood cells (small black arrowheads).
e-48 hr after doing CAA. 17 vs 14 interacting ampullae, respectively. 7 PORs (arrowheads) were all produced by
colony 10, but some were not yeat well developed. In genotype 10, the upper peripheral ampullae still had the
yellow color. f-Two days thereafter. All former PORs were fully developed and no more were added. Ampullae
were cleared of yellow color. g-h-4 days later. Some of the large necrotic lesions were diffused and dispersed
(arrowheads), forming a black line along the contact area between the two genotypes. Interacting ampullae of
both colonies (predominantly of genotype 10) retreated, leaving behind degenerated ampullae and masses of
dying cells (small black arrowheads). 1-5 days thereafter. Ampullae of both genotypes reciprocally retreated,
leaving degenerating areas. Scale of bars: a-g, i=l mm. h=0.25 mm.
few days (usually within 48 hr), other ampullae enhanced by the “retreat growth phenomenon”
continued to interact for longer periods, but with- _[14], where fewer buds than zooids per generation
out the formation of any more PORs (Fig. 1d, g-i). were developed in the contact area, resulting in a
It was also evident in many cases that during POR directional colony growth form, away from in-
development and thereafter, ampullae of one or _ teracting zones (Fig. 1b-d).
both colonies in a pair withdrew from the contact
areas, leaving a bare tunic which gradually deterio- Pairwise allorecognition assays
rated (Fig. 1d, h, i). This process was sometimes We examined 14 Japanese B. schlosseri colonies
986 B. RINKEVICH AND Y. SAITO
in a pairwise allorecognition panel of all 91 com-
binations. Fusions were recorded in 11 (12.1%)
allogeneic assays (Fig. 2a, striped squares) as well
as in all the controls, the isogeneic combinations
(Fig. 2a, striped triangles). An Fu/HC chart for
the studied 14 genotypes (Fig. 2b) revealed 18 to
19 different fusibility alleles. Genotypes 11 and 2
are clearly distinguished from each other since
they possess different color morphs. However,
according to the predictions of the fusibility model
(legend to Fig. 2b), they share in common either
one or both Fu/HC alleles. Twelve out of the
maximum 19 Fu/HC alleles (63.2%) are assigned
to only one colony each, while 5 alleles (F, I, J, P,
R) (26.3%) occurred twice, and 2 alleles (M, N)
(10.5%) occurred three times.
N | {| {|
Colony Allelic combination Colony Allelic combination | | | | |
ABCDEFGHIJK]| "° LMNOPQRS me
Ng y
Fic. 2. A pairwise mer panel between 14 Japanese
B. schlosseri colonies (A) and their Fu/HC allelic
combination chart (B). A-Each rejecting CAA set
is marked by a blank square, fusible pair by a striped
square. All isografts resulted in fusions (striped
triangles). B-The chart depicting predictions for a
single Mendelian locus model of partial genetic
matching for histocompatibility [2, 4-6], an exclu-
sive heterozygosity of the Fu/HC locus in wild
genotypes [2, 12], and a codominant expression by
Fu/HC alleles [2, 4-6]. Colony 11 is marked also by
allele E in brackets to note that it can share with
colony 2 one or both alleles on the Fu/HC ha-
plotype.
DISCUSSION
Intrapopulation allorejection processes in B.
schlosseri from Mutsu Bay, Japan,
are of like
pattern as in the populations from the Venetian
lagoon, from the Mediterranean coast of Israel and
from the Monterey-Santa Barbara areas [6, 9, 10—
15]. The only difference which we could detect
which characterized the Japanese population from
all the above is the marked accumulation of yellow
colored cell aggregations in the tips of interacting
ampullae. Therefore, out of the 5 studied B.
schlosseri populations up to date, only the Woods
Hole population differs significantly from all the
others when comparing between the effector
mechanisms which are expressed during intra- or
interpopulation interactions [11, 12, 16]. Since B.
schlosseri is found in many additional localities
around the world [7, 8, 11], further study is
required to find whether the Woods Hole popula-
tion possesses a unique variation of alloresponses
within this taxon.
Different populations of B. schlosseri are prob-
ably characterized by a highly polymorphic pattern
of alloreactivity. In the Mutsu Bay population,
sampling from a very small area of 5 by 5m
revealed only 12.1% of fusions. In the Woods
Hole population, two different studies have found
6.2% of fusions out of 1,262 contiguous borders of
colonies [17] and 4.2% fusions out of 500 pairs of
colonies collected randomly along a 20 m transect
[18]. Similar low percentage numbers of fusions
were obtained from the Israeli B.
population (Porat and Rinkevich, unpublished).
These studies and others [19] reflect the existence
of approximately 100 histocompatibility alleles in
each studied B. schlosseri population. In Botryllus
primigenus, pairwise fusibility tests revealed a
similar low percentage of fusions [5, 20, 21].
Botrylloides fuscus, on the other hand, panels of
pairwise combinations between colonies which
were collected from 3 localities, 1-3 km apart,
resulted in 64.0-74.0% of fusions. This indicates a
much lower polymorphism on the Fu/HC locus of
schlosseri
this species [22].
The 14 genotypes from Mutsu Bay which were
collected randomly from a very small site possess
18 to 19 alleles on their fusibility locus, of which at
least 2 are commonly shared with the Monterey,
CA, population (colonies 5, 9, 11 fused with
Monterey colonies [16]). Based on the results of
12.1% fusions, we may estimate [23] the numbers
Allorecognition in Japanese Botryllus
of alleles on the Fu/HC haplotype in the Mutsu
Bay population as approximately 32. However, it
is clear that this estimation is minimal and that our
sample does not represent the probable higher
number of alleles residing in the Fu/HC locus of
this population, since colonies were collected from
a very limited (5x5 m) area. Moreover, calcula-
tions are based on the prediction that all the alleles
on the fusibility locus are equally frequent [23],
which is not the case of the present study (Fig. 2b).
This skews the figure for the number of histocom-
patibility alleles and also marks the difficulty of
using CAAs as the only implementation for asses-
sing polymorphism on the Fu/HC haplotype.
Some of these colonies responded to intrapopula-
tion allogeneic encounters by the “retreat growth
phenomenon” [14]. In assays resulting in fusion,
one of the partners in the chimera was usually
resorbed [3, 9, 13], a result which was recorded
before in other B. schlosseri populations as well.
These outcomes suggest that botryllid ascidians
have highly complex systems of effector mechan-
isms, all controlled by a variety of histocompatibil-
ity genes.
ACKNOWLEDGEMENTS
Thanks are due to Dr. Numakunai for helping in
colony collection and to T. Ishii and A. Dasai for general
assistance. The staff of the Shimoda Marine Research
Center is acknowledged for their hospitality and assist-
ance. This study was supported by a grant from S. Price,
North American Friends of IOLR, by a JSPS Fellowship
for Research in Japan (B.R.), and by a Grant-in-Aid for
Scientific Research from the Ministry of Education,
Science and Culture of Japan, nos. 03455008 and
01304007 (Y.S.).
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B. RINKEVICH AND
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ZOOLOGICAL SCIENCE 9: 989-994 (1992)
© 1992 Zoological Society of Japan
Allogeneic Responses between Three Remote Populations
of the Cosmopolitan Ascidian Botryllus schlosseri’
BarucuH RINKEVICH~, MICHAL SHAPIRA~, IRVING L. WEISSMAN”
and YASUNORI SaITo*
National Institute of Oceanography, Tel Shikmona P.O. Box 8030, Haifa
31080, Israel, Howard Hughes Medical Institute, Stanford University
Medical Center, Stanford, CA 94305, USA, +Shimoda Marine
Research Center, University of Tsukuba, 5-10-1
Shimoda-shi, Shizuoka 415, Japan
ABSTRACT— Colony allorecognition assays (CAAs) were performed between colonies of the world-
wide distributed tunicate Botryllus schlosseri, sampled from the Mediterranean coast of Israel (Is), from
Monterey, California (Mon) and from Mutsu Bay, Japan (Ja). While all 48 Is vs Ja CAAs resulted in
nonfusion responses, unexpectedly, 4.4% of the 45 Is vs Mon pairs and 12.0% of the 25 Ja vs Mon assays
ended in colony fusions. Allogeneic effector mechanisms in all 3 populations were similar, except for
the Ja population which was characterized additionally by the appearance of masses of bright yellow
blood cells gathered in the tips of interacting ampullae. A total of 201 multiple CAAs on 24 Is vs Mon,
22 Is vs Ja and 21 Ja vs Mon rejecting pairs did not show an allospecific memory in the rejection
phenomenon. Results are discussed in view of the accumulated data on allogeneic responses in 5 remote
populations of B. schlosseri.
INTRODUCTION
Botryllus schlosseri, a world-wide distributed
colonial ascidian, is a common member of shallow
water hard bottom communities. This species is
found in European waters, Japan, the eastern and
western coasts of North America, Australia, Hong
Kong, and in many other localities [1-4]. This
species, most likely of Mediterranean Sea origin,
became a cosmopolitan species probably by ances-
tral colonies which attached to ship bottoms and
were introduced into new localities.
Like other botryllid ascidians [5, 6], B. schlosseri
colonies show colony specificity resulting either in
vascular anastomosis (fusion) between separate
parts of the same colony or between two compati-
ble colonies, or in rejection between non-
compatible colonies [7-10]. This histocompatibil-
Accepted July 1, 1992
Received April 16, 1992
' Contribution number 542 from the Shimoda Marine
Research Center.
* To whom all correspondence should be addressed.
ity discrimination is controlled by a single gene
haplotype (termed the tunicate’s fusibility-
histocompatibility locus, Fu/HC [11]) with multi-
ple codominantly expressed alleles [6-8]. Two
colonies sharing no alleles at the Fu/HC locus will
reject each other, whereas colonies sharing in
common at least one allele on this locus will fuse
upon direct contact [5-8].
When studying allogeneic reactions between B.
schlosseri colonies collected from Monterey, Cali-
fornia (Pacific Ocean) and from Woods Hole,
Massachusetts (Atlantic Ocean), Boyd et al. [3]
pointed to an interesting result that rejections were
usually confined within the tunic and the peripher-
al ampullae (sausage-like termini of blood vessels)
of only Woods Hole (WH) colonies. Four diffe-
rent types of rejections were developed by only
WH ampullae which included blood cell infiltra-
tion, haemorrhage formation, retraction and
ampullae amputation [4]. More intriguing is the
result that rejection patterns are somehow diffe-
rent in each one of the two tested North American
B. schlosseri populations [3, 4].
990) B. RINKEVICH
Here we further analyze allorecognition re-
sponses between B. schlosseri colonies from 3
extremely separated populations: an eastern
Mediterranean population (from along the coast of
Israel), an eastern Pacific population (from Mon-
terey, CA) and a western Pacific/Japan Sea
population (from Mutsu Bay, Japan). Previous
intrapopulational allogeneic assays revealed that
rejection patterns are similar in these 3 popula-
tions [3, 4, 10, 12, 13]; in short, immediately after
the first tunic-tunic contact was established be-
tween interacting colonies, tips of the marginal
ampullae actively extended in a tip-tip orientation.
Rejections were documented without ampullae
penetration into the tunic matrix of the opposite
colony, and without true matrix fusion.
MATERIALS AND METHODS
We used laboratory reared B. schlosseri colonies
from Israel (Is) and Monterey, CA (Mon), and
wild colonies from Mutsu Bay, Japan (Ja). Origi-
nally wild Mon colonies were collected from Mon-
terey marina and were shipped to the National
Institute of Oceanography, Haifa, Israel, in cool
condition, where they were assayed against col-
onies of the Israeli population. Wild Mediterra-
nean colonies were collected from 3 locations
along the Israeli coast (Tel-Shikmona, Caesarea,
Michmoret). Is and Mon colonies were kept in the
laboratory [3, 12, 14], and offspring were collected
and reared as described [3, 14, 15]. Assays were
performed mainly on reared offspring. Ja colonies
were collected in Mutsu Bay (Aomori, Aomori
Prefecture) and shipped to Shimoda Marine Re-
search Center where they were reared on glass
slides in a wooden culture box immersed in Nabeta
Bay, Shimoda. Is and Mon cultures were shipped
to Shimoda where they were maintained in 17-liter
standing seawater tanks until assayed against Ja
colonies. Colony allorecognition assays (CAAs)
and observations were performed as described [4,
10], and secondary and tertiary tests of CAAs were
performed as previously [4, 16].
, M. SHAPIRA ef al.
RESULTS
Interpopulational allogeneic reactions
We performed 45 CAA pairs of Is vs Mon
colonies, 48 CAAs of Is vs Ja pairs, and 25 CAAs
of Ja vs Mon. While all allorecognition assays of Is
vs Ja resulted in nonfusion reactions (either in
rejections or in indifference [16]), unexpectedly we
observed 2 cases of vascular fusions in Is vs Mon
pairs (4.4%) and 3 fusible assays in interacting Ja
vs Mon pairs (12.0%).
Necrotic lesions (POR=points of rejection)
were developed in assays of the three interpopula-
tional combinations (Fig. la-i) in a similar way as
they were established in _ intrapopulational
allogeneic assays [3, 4, 10, 12, 13, 16]. The only
significant difference in the allorecognition mor-
phology between colonies of the 3 tested popula-
tions was the accumulation of bright yellow blood
cells (morula cells; Tertakover and Rinkevich,
unpublished) which usually gathered in masses
within the tips of interacting ampullae of Ja col-
onies (this was recorded as well in Ja vs Ja com-
binations [13]). These cells disappeared after the
formation of a full set of PORs. PORs were
produced by colonies belonging to the 3 popula-
tions; however, in Ja vs Mon and Ja vs Is combina-
tions, the peripheral ampullae of Ja genotypes
were extended more actively towards those of their
confrere colonies, and more cases were recorded
where PORs were developed by Ja ampullae alone
(in 50.5% of nonfusible Ja vs Mon cases; Fig. 1b-d,
and in 60.0% in Is vs Ja pairs; Fig. la). Cases
where PORs developed by only Mon or Is when
confronted with Ja colonies were much lower
(18.2% and 20.8%, respectively). We observed up
to 11 PORs per assay although up to several tens of
ampullae interacted reciprocally.
Three out of the 4 different characteristic types
of PORs [4], including haemorrhages formation
(Fig. la-f), ampullae amputation (Fig. 1d, f-h) and
formation of an “ampulla POR” were recorded.
Withdrawal of interacting ampullae from contact
areas was another characteristic outcome of inter-
populational allogeneic interactions (Fig. 1b-d).
Ampullae regression appeared in colonies of the 3
tested populations, and usually started within 24 hr
CAAs between Botryllus Populations 991
“ah
a: 5 days after Ja (left)
Fic. 1.
ampullae produced 2 large and diffused PORs, which resulted through ampullar bleeding (arrowheads). Both
previous bleeding ampullae remained intact near PORs (small arrowheads). b: 48 hr after first ampulla-ampulla
contacts. One of the Ja (left) ampullae formed an “ampullar POR” [4] and was amputated and formed a large
POR (arrowhead). Another ampulla formed a typical POR through bleeding (small arrowhead). Zooids of both
partners retreated up to 2mm from the previously interacting site. c-d: 24 and 48 hr, respectively, after first
ampulla-ampulla contact was established. Initial Ja (right) ampullar bleeding, which started with 4 PORs (c,
arrowheads), progressed by the formation of the Sth POR (d, arrowheads) and by ampullae amputation (small
arrowheads). Zooids of both colonies and some of the Mon ampullae retreated from the contact zone. e-i: A case
where Is (left) ampullae were amputated after developing PORs. e: 24 hr after CAA, 3 PORs (arrowheads). f:
24 hr later, 6 PORs (arrowheads), amputation of Is blood vessels leaving only 5 connecting vessels (small
arrowheads) to peripheral ampullae. g: 24 hr thereafter, no more PORs but an additional blood vessel was
amputated (small arrowhead). Ja ampullae started overgrowing the contact area. h: 4 days later, all connecting
vessels were amputated, Ja colony progressed, overgrowing Is ampullae and tunic matrix. 1: 6 days later, Ja
colony was removed to show 6 old PORs (arrowheads) and degenerated tunic of the Is colony. Scale bars: a, c, d
=0.25 mm; b, e-i=0.5 mm.
after the first POR was developed. After ampullar
retreat, the remaining bare tunic gradually de-
generated, forming an empty space between the
interacting colonies.
Multiple colony allogeneic interactions
A total of 201 CAAs were carried out in prim-
ary, secondary and tertiary interaction with 24 Is vs
Mon, 22 Is vs Ja and 21 Ja vs Mon rejecting pairs
(Table 1). Secondary and tertiary interpopulation-
al allogeneic interactions resulted in similar out-
comes as primary interaction, that is, all colonies
continued to express their former mode of
allorecognition responses where again, Ja col-
onies, when confronting either Is or Mon colonies,
developed the yellow color in the tips of interact-
ing ampullae, and were more active in producing
PORs than the colonies of the other populations.
992 B. RINKEVICH, M. SHAPIRA et al.
TaBLE 1. Morphological analyses of colony allorecognition assays carried out in primary, secondary and
tertiary Botryllus schlosseri repeated interpopulational allogeneic reactions
Repeated Is vs Mon CAAs, Repeated Is vs Ja CAAs, Repeated Ja vs Mon CAAs,
in test no.* in test no.* in test no.*
2 P, mi 3 P, Mon 3P,Ja 10d,in | 5P,Ja
SI
4P,rec 6P, rec 2P,Ja 15d,in | 8d,in
|
72, rec 5 P, Is 10d, in Seal eee
2P,Is 6P, mi 2P, Is 3P,Ja |3P,Mon
SP, Is 2 P, Is 7P,Ja 1P,Ja | 4P,Ja
8d,in 1 P, Mon 4P, rec 3P,Ja | SP,Ja
5 P, rec 15 D,in 10d, in 15 d,in |2P, Mon
——!
4P,Ja 10d,in | 4P,Ja
4P,Is 4P, Mon
10 P, mi 1 P, Mon 4P,Ja 4P,Ja | 1P,Ja
&; fy] ee} HR] wl! B]_w] nt] &
=)
2 P, Is 2P, mi
=
2P, Mon 4P, Mon
10d,in| I | 7P,Ja | SP, Ja
1 tm
1P,Ja 2P,Ja |1P,Mon
|
1 P, Is 7P, rec 2P,Ja 3P,Ja | 8P,Ja
2 P, Is 2P, rec 10d, in 2P,Ja | 3P,Ja
2P, Is 6P, Mon 3 P, rec 10d,in |; 2P,Ja
2P,Is 4P, Mon 3P,Ja 2P,Ja | 4P,Ja
Wl Ww] Wi Mm] Ww] ff
4P, Mon 2 P, Mon 3P,Ja 10d,in | 4P,Ja
4P, Mon 5 P, Mon
—
10d, in 1P,Ja | 6P,Ja
1P, Is 3 P, Mon 2P, Is 2P,rec | 8d.in
'1 P, Mon 4P, Mon 4P,Ja 1P,Ja | 4P,mi |
I
3 P, mi 5 P, Is 3 | 10d,in 3 P, rec
it
2 P, Mon 5 | 2P, rec 3 PIs 1P,Ja
| 17d, in 5) | SP. Us LPs Var || 10d, in
7
10 P, Is 2P, Is
4P, Mon 5 | 8P, rec
* Is, Mon and Ja refer to the Israeli, Monterey and Japanese colonies, respectively.
** Results in the first to third tests: P=number of PORs; area where PORs were observed: Is, Mon, Ja, rec
(reciprocal), mi (middle, on the borderlines between the interacting colonies), in (indifference, no POR was
recorded during the period in days [d] specified).
" Int=time interval (in days) from the day on which a full set of PORs was completed until the next assay was
performed. I=acase where a preceding CAA was done immediately after the specified period of indifference or
immediately after the last POR of the former CAA was observed.
CAAs between Botryllus Populations 993
Ja interactions with Is or Mon colonies resulted
also in up to 2-3 times more cases of “indiffer-
ence” within secondary and tertiary assays when
compared to the primary assay of a specific set of
colonies (Table 1). The number of PORs in each
combination of colonies are developed irrespective
of the number of the repeated assay, and their
locations (reciprocally, in the middle between both
colonies or within the tunic of only one partner in
the CAA) were not confined to either one of the
interacting colonies of each specific set of geno-
types (Table 1). The results presented in Table 1
further indicate that secondary and tertiary inter-
populational allogeneic assays done on the same
pairs of colonies (immediately to 17 days after the
last POR of the former CAA was observed; Table
1) do not reveal an allospecific memory in rejec-
tion, for example in an accelerated formation of
PORs and/or augmentation in POR numbers.
These results confirm previous conclusions on
assays done with Mon vs Mon [16] and Mon vs WH
[4] interactions, and further point to the high
variability in directionality of responses/number of
PORs/repertoire of allorecognition responses
characteristic of this species.
DISCUSSION
Recently, 5 B. schlosseri populations were stu-
died for intrapopulation morphology or the gene-
tics of allorecognition: the population from the
Venetian Lagoon, Italy [7, 17], from the
Mediterranean coast of Israel [12], from Woods
Hole, MA, Atlantic Ocean [3, 4, 8], from Mon-
terey and Santa Barbara, CA, eastern Pacific
Ocean [3, 4, 10, 16, 18], and from Mutsu Bay,
western Pacific Ocean/Japan Sea [13]. The results
of these studies elucidate the capacity for distinc-
tion of colony which is manifested by either vascu-
lar fusion or the formation of necrotic lesions when
two different genotypes meet each other through
their peripheral ampullae. In 4 of the above
studied B. schlosseri populations (except the WH
population [3, 4]), tips of marginal ampullae of
paired noncompatible encounters actively ex-
tended towards each other in a tip-tip orientation,
without forming true tunic-matrix fusion. This
continued in the production of PORs without
penetration of ampullae into the tunic of the
confrere genotype. In WH noncompatible pairs,
however, the cortical layers of both tunics usually
become enmeshed and are dissolved in limited
areas near the ampullar tips. This results in
reciprocal penetration of ampullae into the tunic of
the facing colony. On the other hand, all other B.
schlosseri allospecific phenomena, including the
morphology of PORs, are similar to all 5 studied
populations, except for the intensive aggregations
of yellow blood cells in tips of interacting ampul-
lae, which is also characteristic of Ja intrapopula-
tional interactions [13].
Four interpopulation combinations were studied
until now, including the WH vs Mon [3, 4] and Ja
vs Mon, Is vs Ja, Is vs Mon interactions (this
study). We [3, 4 and unpubl.] established more
than 150 WH vs Mon CAAs, which resulted in
zero fusions, similarly to the 48 Is vs Ja CAAs
studied here. It is therefore very surprising that
fusions were obtained in high proportions in Is vs
Mon interactions (4.4%) and Ja vs Mon pairs
(12.0%). In the 25 CAAs of Ja vs Mon, we used 25
Mon and only 11 Ja genotypes, which revealed 3
fusions (done with Ja colonies Nos. 5, 9, 11 [13]).
The allelic pattern of the Fu/HC locus of these 3
fusible Ja genotypes [13] reveals that at least 2-3
alleles on the fusibility haplotype are common to
both Ja and Mon populations. Therefore, it is not
only that these three extremely separated popula-
tions (eastern Mediterranean vs eastern Pacific
Ocean and eastern Pacific Ocean vs Japan Sea,
respectively) belong to the same cosmopolitan
species, namely B. schlosseri (as was concluded
when comparing WH vs Mon populations [3]), but
also these populations are found to possess the
same histocompatibility alleles in each population-
al genetic pool (from a pool of a size of at least 100
allorecognition alleles [9, 19]). This is probably
the reason for the high percentage of interpopula-
tional fusions recorded here.
The results of the present study and of earlier
studies on B. schlosseri interpopulation specific
responses [3, 4] indicate that there are some differ-
ences in allogeneic responses (ampullae penetra-
tions into confronting tunic matrices and the
gathering of yellow pigment cells in tips of interact-
ing ampullae). These responses appear to be
994
characteristic of distinct populations. Additional
studies on other B. schlosseri populations may
elucidate the repertoire of alloresponses character-
istic of this cosmopolitan species.
ACKNOWLEDGMENTS
We thank the staff of the Hopkins Marine Station,
Pacific Grove, and Shimoda Marine Research Center for
the warm hospitality and assistance in conducting this
study. The Is vs Mon part was supported by a Tobias
Landau Fellowship in Marine Biology (B.R.) and by a
grant from the U.S.-Israel Binational Science Founda-
tion (B.R. and I.L.W.). The Ja vs Mon and the Is vs Ja
studies were supported by a JSPS Fellowship for Re-
search in Japan (B.R.) and by a Grant in Aid for
Scientific Research from the Ministry of Education,
Science and Culture of Japan, nos. 01304007 and
03455008 (Y.S.).
REFERENCES
1 Berrill, N. J. (1950) The Tunicata. Ray Society,
London.
2 Tokioka, T. (1953) Ascidians of Sagami Bay. Iwa-
nami-shoten, Tokyo.
3 Boyd, H. C., Weissman, I. L. and Saito, Y. (1990)
Morphologic and genetic verification that Monterey
Botryllus and Woods Hole Botryllus are the same
species. Biol. Bull., 178: 239-250.
4 Rinkevich, B. and Weissman, I. L. (1991) Interpo-
pulational allogeneic reactions in the colonial pro-
tochordate Botryllus schlosseri. Int. Immun., 3:
1265-1272.
5 Taneda, Y., Saito, Y. and Watanabe, H. (1985) Self
or nonself discrimination in ascidians. Zool. Sci., 2:
433-442.
6 Oka, H. and Watanabe, H. (1960) Problems of
colony specificity in compound ascidians. Bull. Mar.
Biol. Stn. Asamushi, 10: 153-155.
7 Sabbadin, A. (1962) La basi genetiche della capaci-
ta di fusione fra colonie in Botryllus schlosseri
(Ascidiancea). Rend. Accad. Lincei, 32: 1031-1035.
8 Scofield, V. L., Schlumpberger, J. M., West, L. A.
and Weissman, I. L. (1982) Protochordate
11
12
B. RINKEvicH, M. SHAPIRA ef al.
allorecognition is controlled by an MHC-like gene
system. Nature, 295: 499-502.
Scofield, V. L. and Nagashima, L. S. (1983) Mor-
phology and genetics of rejection reactions between
oozooids from the tunicate Borryllus schlosseri.
Biol. Bull., 165: 733-744.
Rinkevich, B. and Weissman, I. L. (1988) Retreat
growth in the ascidian Botryllus schlosseri: a con-
sequence of nonself recognition. In “Invertebrate
Historecognition”. Ed. by R. K. Grosberg, D.
Hedgecock and K. Nelson. Plenum, New York, pp.
93-109.
Weissman, I. L., Saito, Y. and Rinkevich, B. (1990)
Allorecognition histocompatibility in a protochor-
date species: is the relationship to MHC semantic or
structural? Immun. Rev., 113: 227-241.
Lilker-Levav, T. (1992) Allogeneic responses in
Botryllus schlosseri and Botrylloides leachi from the
Mediterranean coast of Israel. M. Sc. Dissertation,
Tel-Aviv University (with English summary).
Rinkevich, B. and Saito, Y. (1992) Self-nonself
recognition in the colonial protochordate Botryllus
schlosseri from Mutsu Bay, Japan. Zool. Sci., 9:
983-988.
Boyd, H. C., Brown, S. K., Harp, J. A. and
Weissman, I. L. (1986) Growth and sexual matura-
tion of laboratory cultured Montery Botryllus
schlosseri. Biol. Bull., 170: 91-109.
Rinkevich, B. and Weissman, I. L. (1987) The fate
of Botryllus (Ascidiacea) larvae cosettled with
parental colonies: beneficial or deleterious consequ-
ences? Biol. Bull., 173: 474-488.
Rinkevich, B. and Weissman, I. L. (1992) Incidents
of rejection and indifference in Fu/HC incompatible
protochordate colonies. J. Exp. Zool., 263: 105-
111.
Sabbadin, A. and Astorri, C. (1988) Chimeras and
histocompatibility in the colonial ascidian Botryllus
schlosseri. Dev. Comp. Immun., 12: 737-747.
Rinkevich, B. and Weissman, I. L. (1992) Chimeras
vs genetically homogeneous individuals: potential
fitness costs and benefits. Oikos, 63: 119-124.
Grosberg, R. K. and Quinn, J. F. (1986) The
genetic control and consequences of kin recognition
by the larvae of a colonial marine invertebrate.
Nature, 322: 456-459.
ZOOLOGICAL SCIENCE 9: 995-1000 (1992)
Antibody Production in the Goat: Immunokinetics and Epitope
Specificity Using a Glycoprotein Immunogen
HEATHER Fasry and Jerry L. Hepricx!
Department of Biochemistry and Biophysics, University of California,
Davis, California 95616, USA
ABSTRACT—We have developed methods for the efficient production of antibodies in goats using a
glycoprotein purified from the eggs of the frog Xenopus laevis. When emulsified with complete Freund’s
adjuvant, the amount of immunogen required to elicit high titer antiserum was much less than
commonly used (50 ug as opposed to mg quantities). A single immunizing injection produced a
maximum antibody response and secondary immunizations were not beneficial. The IgG was directed
toward the carbohydrate moiety when the glycoprotein was used as immunogen, although the protein
moiety was immunogenic as shown by the use of the deglycosylated polypeptide. Goats immunized with
glycosylated immunogen all produced maximum titer antisera from 35 to 45 days postimmunization. In
contrast, maximum titer in goats injected with deglycosylated polypeptide was reached at 26 days
postimmunization. We anticipate that the immunization methods reported here will improve the
usefulness of the goat for preparing polyclonal antibodies.
© 1992 Zoological Society of Japan
INTRODUCTION
Methods for producing polyclonal antibodies in
laboratory rodents, such as mice and rabbits, are
experimentally well defined and commonly used.
Although goats are particularly useful for the
production of antibodies because large amounts of
antiserum can easily be collected, experimentally
determined methods for immunization have not
been published. Immunizing procedures for goats
and sheep are usually extrapolated from those
designed for laboratory rodents. For example, it
has been stated that the amount of antigen neces-
sary to stimulate production of antibodies is appro-
ximately proportional to body weight. That is, if
100 4g of protein is optimal for a rabbit, 5-10 mg is
required for a goat [1]. Secondary immunization
(boosting) is also thought to be necessary to pro-
duce maximum titer antisera. Although these
extrapolated methods are currently used [2-4],
there is an acknowledged need for a systematic
study of the immune response in the goat [1, 5].
Accepted July 13, 1992
Received June 4, 1992
' To whom correspondence should be addressed.
The purpose of the research reported here was
to define methods for the most efficient production
of polyclonal antibodies in goats for our purposes
i.e. the minimal amount of immunogen required to
produce a maximal antibody response in a mini-
mum amount of time using a single immunizing
injection. We used as our immunogen a presum-
ably antigenically foreign purified glycoprotein,
the Xenopus laevis cortical granule lectin. The
time required to produce maximum IgG and IgM
titers as a function of immunization dose and
effects of secondary immunization on titer were
determined. We also characterized the epitope
specificity of the antibodies produced to the gly-
cosylated and deglycosylated forms of the lectin
and their macromolecular and tissue specificity.
MATERIALS AND METHODS
Antigen preparation
Xenopus laevis cortical granule lectin (CGL)
was purified from eggs as described [6].
Trifluoromethane sulfonic acid was used to degly-
cosylate the CGL [7]. Xenopus laevis egg en-
velopes were prepared as previously described [8].
996 H. Fasry AND J. L. HEprRIcKk
Protein concentrations were determined using the
bicinchoninic acid method with bovine serum albu-
min as a standard [9].
Immunization procedure
The dairy goats used were does or wethers of the
French Alpine and Nubian breeds or cross breds.
Goats’ ages were variable, ranging from less than
one year to 10. No tissue necrosis was observed in
any of the animals at the sites used for primary or
secondary immunizations.
After dissolving the antigen in 1 ml Tris buffered
saline (10 mM Tris-Cl, 150 mM NaCl, pH 7.5), an
emulsion was made with an equal volume of
complete Freund’s adjuvant (Difco Laboratories,
Detroit MI). Double hubbed needles attached to
glass syringes were used to make the emulsion as
described by Herbert [10]. One intramuscular
injection was made between the clavicle and the
base of the neck. The first boost immunization, if
made, was prepared similarly except that incom-
plete Freund’s adjuvant was used. These boost
injections were generally given in the opposite side
of the neck. Immunogen for the second boost
injection was dissolved in 10 mM phosphate buf-
fered saline, pH 7.4 and injected subcutaneously.
Blood collection and titer determination
Blood was collected by jugular venipuncture
into evacuated untreated tubes (Vacutainer,
Rutherford NJ) and stored at room temperature
for approximately 6 hr to allow clot formation.
After refrigeration overnight at 4°C, the clotted
blood was centrifuged at 10,000 x g for 30 min, the
serum decanted and frozen at —20°C until use.
For large scale collection of blood, we routinely
collected 250 ml and typically recovered 65% of
the volume as serum.
A kinetic ELISA (enzyme-linked immunosor-
bant assay) was used to determine the titer of
serum samples. Disposable ELISA plates (‘Easy
Wash’, Corning Glass Works, Corning NY) were
coated overnight at 4°C with 15 ng of antigen in 50
wl of buffer (100mM NasCO;3, pH9.5). After
washing with TBST (10 mM Tris, 150 mM NaCl,
0.1% Tween 20), 100 «1 of serial 1:3 dilutions of
serum (with TBST) were applied to the wells. For
IgM titer determination, IgG was removed from
serum samples using a protein G kit (Quik-Sep
IgM, Isolab Inc., Akron OH). After incubation at
room temperature for 1 hr, plates were washed
and 100 wl of a horseradish peroxidase conjugated
secondary antibody was added for lhr. Either
rabbit anti-goat IgG (Fc) or rabbit anti-sheep IgM
(uw chain) (both from Organon Teknika Corpora-
tion, West Chester PA) was used as the secondary
antibody. Plates were developed by the addition
of substrate [50 mM citric acid, pH 4.0, 0.60 mM
2,2’-azino bis(3-ethylbenzthiazoline-6-sulfonic
acid), 0.08% v/v H2O;] and the absorbance at 405
nm was measured every 8 sec for 2 min using a
kinetic microplate reader (Molecular Devices,
Menlo Park CA). The velocity, 4A49;/minute,
was plotted against serum dilution. We defined the
antibody titer of antiserum as the inverse of the
serum dilution corresponding to half maximal
velocity.
To confirm that the reported titers represent
immunologically specific binding, a number of
controls were tested: 1) In the absence of antigen,
insignificant amounts of primary antiserum bound
to the ELISA wells. 2) The secondary antisera was
tested for nonspecific binding both to the antigen
and to the ELISA plate. 3) Finally, coincubation
of purified soluble CGL with anti-CGL serum
abolished serum binding to the CGL immobilized
on the ELISA plate.
Preparation of tissue extracts and analysis of serum
specificity
Aqueous extracts were made from fresh Xeno-
pus laevis or mouse tissue by crushing diced organs
between two frosted glass microscope slides in
distilled water. Cellular debris was removed by
low speed centrifugtion. The proteins in the
supernatant solution were separated by reduced
SDS-PAGE [11] and electroblotted onto ni-
trocellulose [12]. Membranes were probed with
antiserum essentially as described for an ELISA,
except that the developing solution contained 50
ml 10mM Tris, 150mM NaCl pH 7.4, 30 mg 4-
Chloro-1-Napthol/10 ml methanol and 50 «l 50%
(w/w) H3Q>.
Antibody Production in the Goat 997
RESULTS
Immunokinetics of IgG production as a function of
immunogen dose
To investigate the minimum amount of im-
munogen necessary to stimulate maximum IgG
production, goats were immunized with 10 to 1000
pug of CGL. Blood samples were collected twice
weekly and the IgG titer of the serum determined.
Immunization with 50, 100 or 1000 ug of CGL
gave virtually identical immune responses. IgG
production began 3 to 8 days postimmunization
and continued to increase until the maximum titer
was reached 35 to 45 days postimmunization.
Figure 1 illustrates the immune response of the
goat immunized with 50 ~g CGL. Maximum titer
values ranged from 30,000 to 60,000 titer units. In
contrast, immunization with 10 “7g of CGL led toa
protracted immune response which peaked at a
lower titer. For example, on day 42 postimmuniza-
tion, the IgG titer from the three goats immunized
with at least 50 wg CGL was at the maximum,
whereas the titer of the goat immunized with 10 ug
LO@ iver
0 10 20 30 40 60 £460
Days postimmunization
Fic. 1. IgG and IgM response after injection of 50 ug
CGL. ;
was around 20% of maximum and reached a
maximum of 7000 titer units on day 65.
Immunization with 100 or 1000 ug of deglycosy-
lated CGL elicited an immune response of slightly
different kinetics compared to CGL. These two
goats’ immune responses were essentially identi-
cal, producing maximum titer antiserum 26 days
postimmunization (approximately 14 days earlier
than goats immunized with 504g or more of
CGL). In addition, maximum titer values were
lower (4,500—6,000 titer units) than those from
goats immunized with CGL.
Effect of secondary immunization on IgG titer
The two goats initially immunized with 100 or
1000 ng CGL were given boost injections in an
attempt to elicit production of higher titer antisera
than was produced after primary immunization.
At the time of boosting (273 days postimmuniza-
tion), the IgG titer had declined to 3% or 7%
(respectively) of the maximum from primary im-
munization. These animals were boosted on two
occasions with a total of 80 ~g CGL. The first
injection was given intramuscularly in an emulsion
of incomplete Freund’s adjuvant and the second
was injected subcutaneously as a solution. After
each boost injection, the serum titer increased and
reached a plateau, but did not exceed the titer
reached after primary immunization (Fig. 2).
Immunokinetics of IgM production
Given the differences in time to reach maximum
IgG titer upon injection of glycosylated and degly-
cosylated immunogen, we looked for similar dif-
ferences in the kinetics of IgM production. IgG
depleted samples from the goat immunized with 50
ug, CGL were analyzed for IgM content using an
IgM specific secondary antibody. Serum samples
from goats immunized with 100 or 1000 ug of
deglycosylated CGL were similarly analyzed for
IgM content, but without removing IgG.
The IgM titer of the goat immunized with 50 pg
CGL was detectable 8 days postimmunization and
peaked between 19 and 22 days postimmunization.
After day 22, the IgM titer slowly decreased
reaching basal levels about 42 days postimmuniza-
tion (Fig. 1).
The IgM response of both goats immunized with
998
Log titer
Boost with
| 50g
Boost with
80g
290 300 310
H. Fasry AND J. L. Heprick
320 330 340 350 360 370
Days postimmunization
Fic. 2.
IgG response after secondary immunization. Goat initially immunized with 100 ~g CGL. Prior to boost, titer
was 3% of maximum produced following primary immunization.
deglycosylated CGL was initially similar to the
goat injected with SO yg CGL in that IgM
appeared 8 days postimmunization. The IgM
response of the goat immunized with 1000 ug of
deglycosylated CGL, however, seemed acceler-
ated because the IgM titer peaked between 12 and
15 days postimmunization and returned to basal
level 22 days after injection. In contrast, the goat
injected with 100 ug of deglycosylated CGL exhi-
bited kinetics similar to those of the goat immu-
nized with 50 ug of CGL in that maximum IgM
antiserum was produced on day 19 postimmuniza-
tion and the IgM serum levels declined gradually.
Epitope specificity of antibodies generated to CGL
and deglycosylated CGL
Upon testing the macromolecular and tissue
specificity of antisera, we observed marked cross-
reactivity of CGL antiserum with Xenopus laevis
egg envelope glycoproteins. However, the CGL
antiserum was tissue specific as it did not react with
aqueous extracts made from Xenopus laevis skele-
tal muscle, liver, gall bladder, spleen, lung, nerve,
and activated egg cytosol. In addition, it did not
react with extracts from mouse lung, kidney, liver,
heart, or skeletal muscle (data not shown). This
cross reactivity with egg envelope glycoproteins
prompted an investigation of whether carbohy-
drate or protein domains were recognized by the
CGL antibody. When CGL was used as an im-
munogen, approximately 96% of the antiserum
reactivity was directed against the carbohydrate
moiety (Fig. 3). In addition virtually all crossreac-
160
Antigen
CGL
dgCGL
VE*
dgVE*
120
S|
E
SS
<
E 0
Py
a
oO
e)
3S
o
> 40
10}
10° io? 107 10" 10° oom
Serum dilution
Fic. 3. ELISA reactivity of CGL antiserum. Reactivity
of anti-CGL serum with given antigens. w CGL;
VE*%, vitelline egg envelopes derived from activated,
dejellied eggs; @ dgVE*, deglycosylated vitelline
egg envelopes; () Preimmune serum; dgCGL, degly-
cosylated CGL.
Antibody Production in the Goat 999
tivity with egg envelope glycoproteins was due to
shared carbohydrate residues, as deglycosylation
rendered envelope glycoproteins unreactive to the
CGL antiserum (Fig. 3). The deglycosylated CGL
antiserum showed no significant crossreactivity
with egg envelope glycoproteins, thereby indicat-
ing that the CGL and envelope protein moieties
were non-homologous (as expected from the li-
mited amount of amino acid sequence data avail-
able on these glycoproteins [13, 14, H. Fabry, M.
Oda, and J. Hedrick, unpublished observations]).
DISCUSSION
Although goats are commonly used to produce
commercial secondary antisera, rabbits and mice
are used more often for production of primary
antisera both in the research laboratory as well as
for commerce. Part of this preference likely stems
from the belief that milligram quantities of antigen
are required for immunization of larger animals.
From our observations, only small amounts of
immunogen are required for the goat. We
observed no differences in the kinetics or magni-
tude of the IgG response when goats were immu-
nized with as little as 50 ~g or as much as 1000 ng
of CGL. These goats all produced maximum titer
antisera (30,000 to 60,000 titer units) 35 to 45 days
postimmunization. Although the IgG response
after immunization with 10 ~g CGL was markedly
slower, useful antisera was eventually produced
(7000 titer units, 65 days postimmunization).
Most immunization schedules call for boost in-
jections at intervals after the primary immuniza-
tion to increase antiserum titer. In addition, it is
often recommended to delay boosting until the
titer in response to primary immunization has
declined to a small percentage of maximum. Our
observations were contrary to this common prac-
tice. We boosted goats initially injected with 100
vg and 1000 ~g CGL on two occasions with a total
of 80 wg of CGL. After each boost, serum titers
increased and reached a plateau, but at no time
was the post boost titer significantly higher than
that produced after primary immunization. Thus,
when adequate amounts of immunogen are used
for the primary immunization using complete
Freund’s adjuvant in the goat, boosting is not
required for generation of maximum titer anti-
serum. However, if relatively small amounts of
immunogen are used (<50 xg), boost injections
may aid in increasing antibody production.
When goats were immunized with CGL, the
dominant immune response was directed against
the carbohydrate or oligosaccharide moiety of the
glycoprotein. Characterization of the sugar
moieties recognized by the CGL antiserum is
currently being investigated and preliminary re-
sults suggest that a modified sialic acid may be the
immunodominant monosaccharide present on the
oligosaccharide sidechains of CGL (H. Fabry, N.
Wardrip, and J. Hedrick, unpublished observa-
tions).
Since the presence of highly immunogenic car-
bohydrate structures on the lectin might lead to an
atypical immune response, we immunized addi-
tional goats with 100 yg and 1000 ug of deglycosy-
lated CGL. The kinetics and magnitude of IgG
production by these goats was not dose dependent,
similar to what was observed when glycosylated
CGL was used. However, the immune response
was accelerated and of lower magnitude than the
goats immunized with the glycosylated lectin.
These differences may reflect genetic variance
between animals or result from the relative im-
munogenecity of the carbohydrate versus the pro-
tein moieties of the CGL.
We have used more than 15 goats in our studies
and all have produced strong immunological re-
sponses to small quantities of Xenopus laevis and
Sous scrofa (pig) protein as well as glycoprotein
immunogens. We feel, therefore, the methods
described in this paper can be successfully applied
to a variety of immunogens, and will improve the
usefulness of the goat for production of primary
antisera.
ACKNOWLEDGMENTS
We thank Dr. Randy Harris for his assistance in
collecting antiserum and Dr. Dan Hardy for helping with
the immunizations. This study was supported in part by a
United States Public Health research grant HD-04906 to
JLH. The care and use of animals followed institutional
and USDA guidelines.
1000
REFERENCES
Dresser, D. W. (1986) Immunization of ex-
perimental animals. In “Handbook of Experimental
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ZOOLOGICAL SCIENCE 9: 1001-1007 (1992)
Appearance of a Nuclear Histone H1 Kinase at the Start of
DNA Synthesis of Regenerating Rat Liver
KouicH! ASAMI
'Division of Biology, National Institute of Radiological Sciences,
Chiba-shi 263, Japan
ABSTRACT—Phosphorylation of histone H1 and elevation of nuclear protein kinase activity occur at
the start of DNA synthesis during regeneration of rat liver. X ray irradiation prior to partial
hepatectomy depressed both elevation of the nuclear protein kinase activity and DNA synthesis. Thus,
the protein kinase seemed to play a role in the start of DNA synthesis, but the property of the enzyme
was not well characterized. The nuclear protein kinase activity was measured with casein or histone H1
as substrate. A protein kinase activity specific for histone H1 was found in the extract from the
regenerating liver nuclei, but not that from the irradiated and partially hepatectomized liver nuclei. On
the other hand, casein kinase activity was found in the extract from the regenerating liver irrespoctive of
prior X ray irradiation. Gel filtration chromatography of the nuclear extract revealed the presence of
the histone H1 kinase and two casein kinases in the regenerating liver. Histone H1 kinase was observed
in the regenerating liver, but not in the non-hepatectomized nor the irradiated and hepatectomized
liver. Two casein kinases were found in the non-hepatectomized liver and partial hepatectomy with or
without prior X irradiation induced a little increase in the activites. The present results indicate that
appearance of the nuclear histone H1 kinase activity is required for phosphorylation of histone H1 and
the start of DNA synthesis in the regenerating liver.
© 1992 Zoological Society of Japan
INTRODUCTION
Hepatocyte in the adult liver does not prolifer-
ate in a normal physiological state. But when a
part of the liver is removed, the rest of the liver
starts proliferating and when the liver mass has
gained the former size the cells return to the
quiescent state. Proliferation of hepatocytes are
stimulated by several growth factors, such as
hepatocyte growth factor or hepatopoietin A [1].
Before the onset of DNA synthesis the stimulated
cells show many responses such as increase in
Na*- H™ exchange, polyamine metabolism, and
amino acid transport [2]. Phosphorylation of his-
tones and HMG proteins which belong to nuclear
non-histone proteins, and synthesis of histones and
HMG (high mobility group) proteins also occur
before and during DNA synthesis, and the en-
zymes of nucleotide motabolism are synthesized at
Accepted July 15, 1992
Received May 8, 1992
' Present address: Department of Biology, Sapporo
Medical College. Sapporo 060, Japan.
the same period [2]. However, causal re-
latiopnships among these events are not clear. A
dose of X rays enough to inhibit the onset of DNA
synthesis inhibited phosphorylation and synthesis
of histone H1, while it failed to inhibit phosphory-
lation and synthesis of HMG proteins [2, 3]. Thus,
phosphorylation of histone H1 seemed to be close-
ly related to the onset of DNA synthesis. Changes
in nuclear protein kinase activity were measured,
therefore, during the prereplicative phase of re-
generation in the liver of partially irradiated rat
[4]. The protein kinase activity increased at the
onset of DNA synthesis and the rise of the activity
was inhibited by X ray irradiation delivered just
before partial hepatectomy [4]. Consequently this
protein kinase seemed to be responsible for phos-
phorylation of histone H1, although its nature was
unknown.
Rat liver nuclei contain several kinds of protein
kinases such as histone H1 kinase [5, 6], casein
kinases [7, 8], protein kinase C [9], Ca/calmodu-
lin-dependent protein kinase [10] and cAMP-
dependent protein kinase [4, 11]. Our previous
1002
results suggested that growth-associated histone
kinase of Langan, a protein kinase specific for
histone H1 found in growing cells [5], was re-
sponsible for the rise of the protein kinase activity
in the nuclei, although participation of other
kinase could not be excluded completely [4].
Therefore, we measured histone H1 kinase and
casein kinase activities in parallel. The present
results indicate that nuclear kinase specific for
histone H1 is responsible for the rise of protein
kinase activity at the onset of DNA synthesis.
MATERIALS AND METHODS
Animals and materials
Male Wistar strain rats, supplied from our
breeding colony, were used between 8 and 10 week
old of age. [y-*’P]ATP was purchased from Amer-
sham, Japan. PMSF (phenylmethylsulfony!
fluoride), a-casein (Sigma), and Combithek (pro-
tein standard set: Boeringer) were used. Histone
H1 was extracted from rat liver according to the
procedures of Sanders and Johns [12], and the acid
soluble fraction was passed through a column of
BioRex 70 (Bio-Rad Lab., USA) [13]. The frac-
tion containing histone H1 was collected, freeze-
dried, and dissolved in water. Fractions containing
mainly histone Hlg were not used. Histone H1
solution, which showed a single band with polyac-
rylamide gel electrophoresis, was divided in small
aliquots and stored at —20°C until use.
X ray irradiation and partial hepatectomy
X ray irradiation and partial hepatectomy of rats
were carried out as described previously [3, 4].
During anesthesia with pentobarbital, rats were
irradiated to their upper abdomen including the
liver with 4.8 Gy of X rays. The condition of
irradiation was 200 kVp and 20mA and X rays
were filtered through 0.5 mm Cu and 0.5 mm Al.
Following irradiation, the median and the left
lateral lobe of the liver were removed. The
non-irradiated hepatectomized
under anesthesia with pentobarbital. The right
lateral and caudate lobes were used in the experi-
rats were also
ments of non-hepatectomized rats, since these
lobes were used in the case of hepatectomized rats.
K. ASAMI
The hepatectomized rats were killed at 24h after
partial hepatectomy, when DNA synthesis started
[3] and the protein kinase activity increased [4].
Preparation of nuclear extract
Isolation of nuclei, essentially according to the
procedure of Blobel and Potter, and preparation
of the nuclear extract were described previously
[4]. Briefly, the nuclei were extracted with 0.4 M
NaCl in buffer A, which consisted of 20mM
TrisHCl pH 7.5, 1mM EDTA pH7.5, 0.1mM
PMSF, SmM_ benzamidine, 2mM_ glycerol-2-
phosphate and 10mM 2-mercaptothanol. After
treatment with BioRex 70, the extract was mixed
with potassium phosphate pH 7.5 and cAMP to
give 0.1M and 1M respectively in order to
separate cAMP-dependent protein kinase [5, 6].
The protein precipitated with ammonium sulfate
between 17.5% and 35% saturation was collected.
The precipitate from one rat (ca. 5g liver) was
dissolved in 0.5 ml buffer A containing 0.4M
NaCl. The extracts thus obtained were dialyzed
overnight against about 50 volume of buffer A
containing 10% glycerol. For resolution with
chromatography the nuclei from 3 rats were com-
bined before extraction and the extracts were used
for gel filtration chromatography immediately af-
ter extraction. Although 0.4M NaCl did not
extract all the kinase activity, the portion of the
extracted activity was not changed much during
regeneration [4]. The protein kinase activities
were hence compared on the nuclear extracts with
0.4 M NaCl in the present experiments.
Column chromatography of the extract
FPLC system (Pharmacia) was used. When
Mono Q column was used to separate the extract,
dialysis of the extract before chromatography
caused aggregation of the proteins and almost all
proteins remained on the top of the Mono Q
column. Therefore, the extract was applied to the
column of Superose 12 immediately after extrac-
tion. The column was equilibrated in 0.4 M NaCl
containing buffer A, and ca. 2 mg of the nuclear
extracts were applied. Fractions of 1 ml were
collected, and the enzyme activity and protein
concentration were determined. Protein was de-
termined according to the procedure of Bensadoun
H1 Kinase in Regenerating Liver Nuclei
and Weinstein [14].
Assay of protein kinase activity
A similar procedure with the previous experi-
ments was used with rat liver histone H1 and
a-casein as substrate [4]. Briefly, the reaction
mixture contained in 0.05 ml; 50 mM TrisHCl pH
7.5, 10mM MgCh, histone H1 (0.5 mg/ml) or
a-casein (0.5 mg/ml), 0.5 mM ATP (containing 1
uCi [y-°P]ATP), 1mM EDTA pH7.5, 1mM
dithiothreitol, and 20mM _ glycerol-2-phosphate.
The reaction was started with addition of the
enzyme. After incubation for 10 min at 37°C, the
reaction was stopped with addition of 0.25 ml 20%
trichloroacetic acid. The precipitate was collected
on a Millipore filter and washed with 10% trichlor-
oacetic acid. After drying in air, radioactivity of
the filter was measured with a liquid scintillation
counter. Since incubation without substante
caused substantial uptake of radioactivity, net up-
take of radioactivity was calculated by subtracting
the values obtained without substrate.
time value was subtracted.
The zero
RESULTS
Normal liver nuclei contain a protein kinase or
kinases and partial hepatectomy causes partial
increase in kinase activity at the time of DNA
synthesis [4]. More than one protein kinase could
be measured simultaneously in the previous ex-
periments, however, since histone mixture used as
the substrate contained histone mixture as well as
non-histone proteins (data not shown). In the
present experiments, therefore, protein kinase
activity was measured with more defined substrate;
rat liver histone H1 and casein. Phosphorylation
due to endogenous substrate was subtracted from
the values with the substrates, and also shown in
the results separately (Table 1). The protein
kinase activity phosphorylating histone H1 was
observed in the extract from regenerating liver
nuclei, while in was not observed in the extract
from the nuclei of the X ray-irradiated and partial-
ly hepatectomized rat (Table 1). On the other
hand, the protein kinase activity phosphorylating
casein was observed in both the hepatectomized
and the irradiated and hepatectomized nuclear
1003
TABLE 1. Protein Kinases in the Nuclear Extract of
Regenerating Liver
Activity (nmol/10 min/mg protein)
Substrates
Non-irradiated X-irradiated
0.63+0.57 P<0.05
histone H1 3.66 £0.93
casein 4.75 + 1.60 3.65 +0.80 NS
(endogenous) 7.14+1.66 Sil se WS NS
The nuclear extract was prepared from the rat at 24
h after partial hepatectomy. Mean of 3 indepen-
dent experiments with SEM.
extract. Phosphorylation of endogenous substrate
was also observed on all the groups measured
(Table 1).
changes in the activity phosphorylating casein or
endogenous substrate. These results indicated
presence of histone H1 kinase distinct from casein
kinase | and 2 and responsible for phosphorylation
of histone H1 at the onset of DNA synthesis in the
regenerating liver nuclei.
To confirm the presence of a protein kinase
more specific to histone H1 than casein in the
nuclei, the nuclear extracts were fractionated with
gel filtration chromatography using Superose 12
column. The nuclear extract dissolved in buffer A
containing 0.4 M NaCl was immediately applied to
Superose 12 column equilibrated in the same
medium. The protein kinase activities of each
fraction was measured in the presence of histone
H1 or casein or without substrate addition (Fig. 1).
Elution profile of proteins did not change much
among the non-hepatectomized, the hepatecto-
mized and the X-irradiated and hepatectomized
liver. The activity phosphorylating endogenous
substrate(s) was found in the fraction eluted at the
void volume and decreased in the later fractions.
Endogenous substrate(s) were probably associated
with its protein kinase in the nuclear extract and
pass through the column in the associated form.
The activity phosphorylating casein was eluted in
Irradiation caused no _ substantial
two peaks, i.e. fraction 10 and 15 as maximal
respectively, while that phosphorylating histone
H1 was maximal in fraction 12. The liver nuclei
have been reported to contain two casein kinases
[7, 8]. The two peaks probably correspond to the
nuclear casein kinase | (fraction 15) and 2 (fraction
ENZYME ACTIVITY (pmol/10 min )
1004
100
10) or NI and NII [7, 8]. The casein kinase activity
after subtraction of the activity without substrate
addition were divided into two parts, i.e. the
fractions 13-17 and the fractions 7-12, and they
were designated here as casein kinase 1 and 2
respectively. The integrated activities are shown in
Table 2. Activities of casein kinases of hepatecto-
mized liver are a little higher than those of non-
hepatectomized liver, but X ray irradiation hardly
affected the activities.
An activity phosphorylating histone H1 was
eluted in the fractions of regenerating liver be-
tween casein kinase 2 and 1. This indicates that
casein kinases and histone H1 kinase are different
enzymes. The nuclear extract from the non-
hepatectomized liver or from the X-irradiated and
hepatectomized liver contained casein kinase 1 and
2, but no histone H1 kinase. The activity
phophorylating endogenous substrate was sub-
tracted from that in the presence of histone H1 for
each fraction. Phosphorylating activities of frac-
tions 8 to 9 or to 11 were lower in the presence of
histone H1 than in its absence, indicating that
histone H1 inhibited the phosphorylation of the
endogenous substrate. The fractions, where the
activity was higher in the preence of histone H1
Fic. 1. Gel filtration chromatography of the nuclear
extracts from rat liver. Superose 12 attached to
FPLC system was used. (A) Partially hepatecto-
mized (2.54 mg protein), (B) partially irradiated and
hepatectomized (2.58 mg protein), and (C) non-
hepatectomized (2.58 mg protein). Histone H1 (.),
6 A 10 12 % IG j@ 20 casein (A) and none (@) were used as substrate.
FRACTION NO. Activity in 25 “1 of the eluate was shown in the
figure. ...., Protein contents.
TABLE 2. Protein Kinases in the Nuclear Extract of Rat Liver
7 xX Now Activities (nmol/10 min/mg protein)
at ae : Ss
cays EXD. HK CKI CKI
Non-HPX = 3 0.58 +0.09 10.87 +£0.78 3.88 + 0.54
HPX — 2 2.31 +0.46" 17.52+4.78 4.74 + 1.42
HPX + 3 0.62 + 0.36 15.30+ 1.38" 4.16+1.33
The nuclear extract from the non-hepatectomized (non-HPX) or the hepatectomized (HPX) rat was used
for FPLC. Protein kinase activities and protein centents of each fraction were measured and summed
up.
HK; histone H1 kinase, CKI and CKII; casein kinase | and 2 respectively. The asterisks indicate
a significant difference from the non-hepatectomized (P<0.05).
H1 Kinase in Regenerating Liver Nuclei
than in its absence, were regarded to contain
histone H1 kinase and their differences are sum-
med up (Table 2). Nuclear histone H1 kinase
activity of the hepatectomized liver was higher
than either the non-hepatectomized or the X ray-
irradiated and hepatectomized liver. Partial in-
activation of the histone H1 kinase however was
noticed on the fractions after chromatography,
since the specific activity after chromatography
was similar to that before chromatography (com-
pare Table 2 with Table 1). The reason of inactiva-
tion is unknown, but instability of growth associ-
ated histone kinase after chromatography was re-
ported [6, 15].
Apparent molecular weight of three protein
kinases were calculated from chromatography of
the standard proteins; ferritin, catalase, aldolase,
bovine serum albumin, egg albumin, chymotrypsi-
nogen A and cytochrome c. Some of the standard
proteins, especially ferritin, showed peakds with
smaller molecular weight after chromatography.
This would be due to depolymerization of the
polymerized form of the proteins in the elution
buffer containing 2-mercaptoethanol. These peaks
with smaller molecular weight were also used for
calculation. The obtained regression line was
log(molecular weight)=5.32—0.137 x (retention
time in min); r=—0.9646. The calculated sizes of
the protein kinases were 381 kDa (casein kinase
2), 92 kDa (histone H1 kinase), and 12 kDa (ca-
sein kinase 1), respectively (data not shown).
The results shown in Figure 1 indicated that
histone H1 inhibited phosphorylation of endoge-
nous substrate by the kinase eluted simultaneous-
ly. Phosphorylation of endogenous substrate and
casein was then measured in the presence of
different concentrations of histone H1, since ca-
sein kinases were the protein kinase found in the
nuclei in the present condition except for histone
H1 kinase (Table 3). Histone H1 inhibited phos-
porylation of casein by casein kinase 1 (fraction 15)
or 2 (fraction 10). Phosphorylating activity of
fraction 8 which contained endogenous substrate
was activated with casein and the activity was
inhibited by histone H1 irrespective of addition of
casein.
1005
TABLE 3. Inhibition by histone H1 of the phos-
phorylating activity of the nuclear extract with or
without casein
histone H1 (mg/ml reaction mixture)
Enzyme Casein
0 0.5 1.0
nmol/10 min/mg protein
Fr. 8 + 5.50 6.56 3,32
= 2.54 1.62 1.18
Fr. 10 ar 10.98 9.41 5.04
= 1.63 1.91 1.33
Fr. 15 3 37.17 17.32 16.73
- 0.80 1.33 1.98
The values with casein are not corrected for *°P
uptake without casein. Fraction (Fr.) 10 and Fr. 15
contain casein kinase 2 and casein kinase | respec-
tively.
DISCUSSION
Phosphorylation of histone H1 is closely related
to the initiation of DNA synthesis of regenerating
liver [2, 3]. However, it is still not known which
enzyme is responsible for the phosphorylation.
Although cAMP-dependent protein kinase was
found in the nuclei [11], the enzyme was not
related with phosphorylation of histone H1 at the
time of DNA synthesis [4] and was removed from
the nuclear extract in the present experiments
through treatment with cAMP and ammonium
sulfate precipitation [5, 6]. Protein kinase C and
Ca** /calmodulin-dependent protein kinase II are
also reported in rat liver nuclei [9, 10], but the
present assay condition was not favorable for the
Ca-dependent kinases, since the assay medium
contained no Ca** but EDTA. The inhibitors of
the protein kinase C did not inhibit the nuclear
kinase [4]. These protein kinases, therefore, will
not be considered further in this article.
The present results show that the nuclear extract
of the regenerating liver contained two kinds of
casein kinases and one histone H1 kinase. The
extract seemed to contain another protein kinase
which was associated with endogenous substrate.
This will be a casein kinase as discussed later.
Two kinds of casein 1 (NI) and 2 (NII), although
the molecular weight of casein kinases in the
present results are not consistent with the reported
1006
values (e.g., 50 and 133 kDa [8] or 35 and 200 kDa
[15] respectively). In the present paper however
they are called casein kinase 1 and 2. Activities of
casein kinases increased a little in the regenerating
liver and X rays enough to inhibit phosphorylation
of histone H1 scarcely affected the increase. A
nuclear non-histone protein, HMG 14 is phos-
phorylated with casein kinase 2 [8, 16] and X rays
do not affect phosphorylation of HMG 14 [3].
These are in consistent with the results that X rays
scarcely affected the activity of casein kinase.
Both casein kinase 1 and 2 are inhibited by
histone H1. An endogenous inhibitor protein of
casein kinase was reported previously, which was a
25kDa protein found in high mobility group
(HMG) protein fraction of rat liver, but it was not
characterized further [17]. However, histone H1
was not removed from their HMG fraction, which
usually contained histone H1, before separation of
the inhibitor [17]. The present results suggest that
this endogenous inhibitor protein of casein kinase
is histone H1. Inhibition by histone H1 of nuclear
casein kinase may have physiological significance
for regulating phosphorylation of nuclear proteins,
although more experiments should be carried out
before concluding histone H1 as a natural inhibitor
of nuclear casein kinase.
A protein kinase is associated with endogenous
substrate and that is the cause of high phosphoryla-
tion rate without added substrate (Table 1). The
enzyme was able to phosphorylate casein (or it was
activated with casein) and it was inhibited by
histone H1. It was reported that casein kinase 2 in
the nuclear extract was associated firmly with high
molecular weight protein such as nucleolin or
progesterone receptor [18, 19]. Hence, the en-
zyme phosphorylating endogenous substrate may
be casein kinase 2.
Histone H1 kinase was found in the regenerating
nuclei, while it was not observed in the non-
hepatectomized nuclei. X irradiation inhibited the
appearence of the histone H1 kinase activity in the
regnerating nuclei. Casein kinases had a very low
affinity for histone H1 as substrate. Therefore, the
nuclear histone H1 kinase will be responsible for
phosphorylation of histone H1 at the time of DNA
synthesis.
K. ASAMI
The present results coincide with the previous
results that the enzyme responsible for phosphory-
lation of histone H1 will be a growth associated
histone kinase of Langan [4]. Apparent molecular
mass of the kinase was 92 kDa. Molecular weight
of growth associated histone kinase is not re-
ported, but that of catalytic subunit is 67,000 [6].
Cyclin-p34°“* protein kinase is specific for histone
H1 and is thought to be the same as growth
associated histone kinase [20, 21]. Association of
p34°4? with cyclin, 62 kDa, will constitute a com-
plex of 96kDa, a similar value to the present
results, although apparent molecular mass of cyc-
lin-p34°"? complex is evaluated as 220 kDa from
chromatography with Superose 12 [21]. Nuclear
localization of the kinase has been shown [22, 23].
Although the cyclin-p34~ play a role in the G2 to
M transition of the cell cycle, the same enzyme or
its homologue is claimed to participate for the G1
to S transition [24-26]. Thus the histone H1 kinase
found in the nuclei will be growth associated
histone kinase or a kinase related to it, which will
play a role in phosphorylation of histone H1 at the
onset of DNA synthesis.
The mechanism how X rays inhibit appearance
of the histone H1 kinase in the nuclei is not known.
It is not known whether X rays affect directly the
appearance of histone H1 kinase in the nuclei.
Instability of growth-associated histone H1 kinase
has been reported [6, 15, 27]. Activity of p34
protein kinase can be controlled by binding of
cyclin which is usually synthesized in each cell
cycle, and the activity can be changed depending
on the phosphorylation state of these two proteins
[28, 29]. On the other hand, synthesis of p34‘?
during the G1 phase of stimulated lymphocytes
was Observed [25]. Hence X rays may suppress
appearance of the histone H1 kinase in the nuclei
by changing the cellular metabolism resulting in
inactivation of the kinase, or by inhibiting de novo
synthesis of protein(s) constituting the kinase.
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ZOOLOGICAL SCIENCE 9: 1009-1015 (1992)
Immunohistochemical Demonstration of Metallothionein in the
Rat Epididymis and Spermatic Cord
TAKANORI SUZUKI, HIDETOSHI YAMANAKA, Kew Suzukt',
Katsuyuki NAKAJIMA~, KAzUSHI KANATANI,
Masami Kimura® and Noriko Oraki‘
Department of Urology, Gunma University, School of Medicine, Maebashi,
'Department of Pathology, Medical Care and Technology, Gunma
University, Maebashi, "Japan Immunoresearch Laboratories
Co., LTD, Takasaki, *Department of Molecular Biology,
Keio University School of Medicine, Tokyo, and
*Central Institute of Experimental Animals,
National Institute of Industrial Health,
Kawasaki, Japan
ABSTRACT—We studied the immunohistochemical localization of metallothionein (MT) in the
epididymis and spermatic cord of male Wistar rats. In the head of the epididymis, no MT
immunoreaction of epithelial cells in the ductuli efferentes or ductus epididymidis was observed, but
basal cells of the ductus epididymidis showed a positive immunoreaction for MT. In the body of the
epididymis, a few epithelial cells in the ductus epididymidis had a positive immunoreaction for MT, but
no positive immunoreaction was observed in the basal cells. In the tail portion near the body, basal cells
in the ductus epididymidis showed a positive immunoreaction for MT while the epithelial cells had a
negative one. In the portion near the spermatic cord, both the epithelial and basal cells in the ductus
epididymidis had a positive immunoreaction for MT. In the spermatic cord, basal cells and some
epithelial cells had a positive immunoreaction for MT. The localization of MT was observed mainly in
the cytoplasm of the cells, with some nuclei of the cells having a positive immunoreaction. Different
immunoreactions for MT were demonstrated in the epithelial and basal cells of the epididymis and
spermatic cord, which suggested that epithelial cells function differently in various portions of the
epididymis.
© 1992 Zoological Society of Japan
INTRODUCTION
Metallothionein (MT) is a known metal-binding
protein with a low molecular weight, approximate-
ly 6,000 MW, and an ability to bind closs II-B
metals, such as zinc and cadmium [1]. It has been
identified in a variety of organs, including the liver,
kidneys, prostate, and testes [1-4]. MT is induced
in response to various physiological stimuli and
endogenous factors [5]. Although the exact func-
tions of MT are not clearly known, it has been
considered to play a role in the detoxication,
Accepted July 2, 1992
Received May 6, 1992
storage, and metabolism of heavy metals [6].
The epithelial cells of the human and rat pros-
tate secrete MT [3, 7, 8] and zinc, an action which
correlates with infertility [9]. Recently, MT was
reported to be localized in the spermatogenic cells
[10, 11], but few studies on MT in the epididymis
have thus far been undertaken. The purpose of the
present study was to demonstrate the localization
of MT in the epididymis and spermatic cord as part
of an investigation of the function of MT as it
relates to sperm.
MATERIALS AND METHODS
Male Wistar rats (10 weeks old) were purchased
1010
from Imai Experimental Animals (Saitama, Japan)
and were anesthetized by ether. The tissues ex-
amined by immunohistochemical staining were
obtained from the epididymis (head, body, and
tail) and spermatic cord.
Preparation of tissues for immunohistochemical
examination
All tissues were fixed in 10% buffered formalin
solution for 48 hours and embedded in paraffin.
Specimens were cut at a thickness of 3 ~m and
mounted on glass slides. Staining was performed
using an avidin-biotin peroxidase
method. Sections were deparaffinized and incu-
bated in 0.1% trypsin solution for 30 minutes at
37°C. They were then immersed in 0.5% (5 mM)
periodic acid solution for 10 minutes in order to
inhibit endogeneous peroxidase, and incubated
with normal goat serum for 20 minutes to block the
non-specific binding sites. Primary antibody was
mixed with a 200-fold concentration of anti-MT-1
and ascaris antigen to inhibit non-specific reaction
to ascaris, because anti-MT-1 antibody was
obtained from rabbit antiserum boosted by MT-1
emulsion coupled to ascaris antigen with glutaral-
dehyde [7]. The slides were layered with primary
antibody for 2 hours at room temperature and
complex
T. Suzuki, H. YAMANAKA ef al.
washed with 0.01M PBS buffer (pH7.2). The
secondary antibody was applied for 1 hour and
sections were again washed with PBS. They were
treated with ABC complex for 30 minutes and
washed with PBS. Sections were submerged in
0.05% diaminobenzidine tetrahydrochloride in
0.05M Tris buffer (pH7.6) to which H>O>
(0.01%) has been added just before use. These
reagents were prepared using a VECTASTAIN
ABC KIT (VECTOR Laboratories, Burlingame,
USA). Subsequent counterstaining was performed
with Mayer’s hematoxylin. The specificity of the
staining reaction was determined in a prior absorp-
tion of anti-MT antibody with pure liver MT
antigen (MT-1 and/or MT-2) (Sigma Chem. Co.,
USA) and omission of primary antibody from the
procedure.
RESULTS
Immunohistochemical control
As shown in Figure 1, the use of primary anti-
body that had been absorbed with pure rabbit liver
MT-1 and MT-2 did not result in any specific
immunoreaction in the epididymal head or tail or
in the spermatic cord. Use of non-immune serum
Fic. 1. Control section of the ductus epididymidis in the tail portion treated with preabsorbed primary antibody.
Immunoreaction for MT was negative in the epithelial and basal cells (x 220).
MT in Rat Epididymis and Spermatic Cord 1011
and PBS did not result in any immunoreaction in _ tained two ducts, the ductuli efferentes and ductus
any sections. epididymidis. No immunoreaction for MT was
observed in the epithelial cells of the ductuli
efferentes (Fig. 2). In the ductus epididymidis,
basal cells showed positive immunoreaction for
Immunohistochemical findings
The head of the epididymis. This portion con-
Fic. 2. Photomicrograph of the ductuli efferentes.. Immunoreaction for MT was negative in the epithelial and basal
cells and in connective tissues (Xx 560).
Fic. 3. Photomicrograph of the ductus epididymidis in the epididymal head. Immunoreaction for MT was found in
the basal cells, but not in the epithelial cells or connective tissues (560).
1012 T. Suzuki, H. YAMANAKA et al.
MT, but epithelial cells and connective tissues The body of the epididymis. A few epithelial
showed a negative one. MT immunoreaction was cells of the ductus epididymidis, about one or two
localized mainly in the cytoplasm and partly inthe cells per one section of the ductus epididymidis,
nuclei of epithelial cells (Fig. 3). showed a positive immunoreaction for MT, but
, b as P : 5 Es a e <. 4 *. s 3
Fic. 4. Photomicrograph of the ductus epididymidis in the epididymal body. Immunoreaction for MT was found
positive in one epithelial cells, and was localized mainly in the cytoplasm. The basal cells, sperm and fluid in the
lumen, and connective tissues were negative (350).
pein ee “IS SET SSS ETE. ED
ere - ——SS GN A Ze
= -
So a
~ ~~ = a a
Be OR OE a a
SG SS ease “a :
2 = -
Fic. 5. Photomicrograph of the ductus epididymidis in the epididymal tail portion near the body. Basal cells had a
positive immunoreaction for MT, but epithelial cells and connective tissues and sperm in the lumen were negative
(x 560).
MT in Rat Epididymis and Spermatic Cord 1013
most epithelial cells were nagative. The basal plasm, which appeared as fine granular immunos-
cells, sperm and fluids in the lumen, and connec- taining (Fig. 4).
tive tissues had no immunoreaction. MT im- The tail of the epididymis. In the portion near
munoreaction was localized mainly in the cyto- the body, basal cells of the ductus epididymidis
y,
]
ite SS
Se
Fic. 6. Photomicrograph of the ductus epididymidis in the epididymal portion near the spermatic cord. Im-
munoreaction for MT was found strongly positive in the epithelial and basal cells, and was localized mainly in the
cytoplasm, especially the apical area, and partly in the nucleus. Connective tissues and sperm in the lumen were
negative (x 560).
Fic. 7. Photomicrograph of the ductus deferens. Immunoreaction for MT was found positive in the basal cells and
some tall epithelial cells. Connective tissues and sperm in the lumen were negative (x 350).
1014
exhibited a positive immunoreaction for MT, but
epithelial cells showed a negative one. The sperm
and fluids in the lumen and the connective tissues
exhibited a negative immunoreaction (Fig. 5). In
the portion near the spermatic cord, the epithelial
and basal cells of the ductus epididymidis showed a
strong positive immunoreaction for MT, which was
localized mainly in the cytoplasm, especially the
apical area, and partly in the nuclei. The staining
in the cytoplasm had a fine granular character.
The sperm and fluids in the lumen and the connec-
tive tissues were negative for immunoreaction
(Fig. 6).
Spermatic cord. Some tall epithelial cells of the
ductus deferens showed a positive MT im-
munoreaction as did many basal cells. The loca-
lization of MT immunoreaction was seen mainly in
the cytoplasm and in some nuclei (Fig. 7). Epithe-
lial cells of the portion near the urethra were
mostly negative. The connective tissues and the
sperm and fluids in the lumen were negative. The
basal cells in the ejaculatory duct also showed a
positive immunoreaction for MT. Connective tis-
sues surrounding the ejaculatory ducts exhibited a
negative immunoreaction.
DISCUSSION
The presence of MT in rat testes was reported by
Nolan and Shaikh [12], and immunohistochemical-
ly demonstrated by Danielson [4] and Nishimura er
al. [10]. MT immunoreaction was observed in the
spermatogenic cells in the seminiferous tubules,
but not in mature sperm in the lumen. Recently,
De et al. [11] reported that MT mRNA which
accumulated after the initial differentiation of
primary spermatocytes was maintained in sperma-
tids, and was shown to be present at low levels in
interstitial, spermatogonial, and matured sperm
cells by Nothern blooting and in situ hybridization.
Therefore, they suggested there being a role for
MT in the process of spermatogenesis. In this
study, matured sperm in the lumen of the ductuli
efferentes, ductus epididymidis, and ductus de-
ferens had a negative immunoreaction for MT,
suggesting that they did not synthesize MT.
MT was identified in the prostatic cells of rats
and humans, and was shown to secrete into the
T. Suzuki, H. YAMANAKA et al.
prostatic fluids [3, 7, 8]. The seminal fluids mainly
consisted of those derived from the testis and
epididymis, the prostate, and the seminal vesicle.
MT is known to bind to zinc, an action which is
reported to correlate with infertility [9, 13].
However, few studies on MT in the epididymis
have thus far been undertaken, and the physiolo-
gical function of MT in the epididymis is unclear.
Nishimura et al. [10] reported that MT im-
munoreaction was observed in a limited number of
epithelial cells and in some basal cells in the ductus
epididymidis of the rat. The present study demon-
strated the differences of immunoreactions for MT
in the various portions of the ductus epididymidis
and spermatic cord. The basal cells had a positive
immunoreaction for MT in the head and tail
portions of the ductus epididymidis, but not in the
body. The epithelial cells had a positive im-
munoreaction in the body and tail, especially in
many of the epithelial cells in the tail portion near
the spermatic cord. The spermatozoa first to leave
the testis are incapable of fertilization and are
described as being immature. The sperm in the tail
of the epididymis are mature and capable of ferti-
lization [14]. The charactristic feature of matura-
tion is a change in the surface of the sperm, as well
as the presence of glycoprotein secreted from the
epididymal epithelial cells which has been reported
to bind to the sperm surface [15]. The principal
cells has numerous cisternae of rough endoplasmic
reticulum and well-developed Golgi apparatus
associated with secretory function, and the MT
localized in the epithelial cells of the tail portion
was suggested to be secreted into the epididymal
fluids [16, 17]. In this study, however, the secre-
tion of MT could not be clearly demonstrated
under light microscopy, and an electron immuno-
histochemical study was though to be necessary to
demonstrate the correlation between the sperm
and MT in the epididymis.
In this study, the localization of MT was
observed mainly in the cytoplasm and partly in the
nuclei. Histologically, MT localization in the nuc-
leus has been reported by Nishimura et al. [10] to
be present in the spermatogonial cells, and in the
prostatic cells of the rat by many investigators [3,
7, 10, 18]. The positive staining of epithelial and
basal cell nuclei might conceivably result from
MT in Rat Epididymis and Spermatic Cord
contamination of nuclear proteins by the MT
transposed during tissue preparation. However,
no nuclei had ever shown a positive immunoreac-
tion in the epididymis, reducing the likelihood of
this explanation. Recently, large molecules con-
taining the nuclear location signal sequence have
been reported capable of being transported into
the nucleus crossing the nuclear envelope of
mammalian cells [19, 20]. This suggests that MT
may also be transported into the nucleus and act as
an enzyme activator by donation of zinc bound to
it.
In summary, the localization of MT and differ-
ences of immunoreaction for MT in the various
portions of the ductus epididymidis were demons-
trated. Many epithelial cells in the tail had a
positive immunoreaction for MT, and MT was
suggested to function in the maturation of sperm.
ACKNOWLEDGMENTS
This work was supported in part by a Grant-in-Aid
from the Ministry of Education, Science and Culture
(02454367), Japan.
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2 Danielson, K. G., Ohi, S. and Huang, P. C. (1982)
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3 Umeyama, T., Saruki, K., Imai, K., Yamanaka, H..,
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5 Bremner, I. and Davies, N. T. (1975) The induction
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Saito, S., Bush, I. M. and Whitmore, W. F. Jr.
(1967) Effects of certain metals and chelating agents
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Nishimura, H., Nishimura, N. and Tohyama, C.
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De, S. W., Enders, G. C. and Andrews, G. K.
(1991) High levels of metallothionein messenger
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Morisawa, M. and Mohri, H. (1972) Heavy metals
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ZOOLOGICAL SCIENCE 9: 1017-1023 (1992)
Analysis of Sex Steroids in Feces of Giant Pandas
Kaoru Kusoxawa!”, Susumu IsHu’, HipeEo Tasmma®
Kazuo Saitou® and Kouk! TANABE>
'? Department of Biology, Waseda University, Shinjuku,
Tokyo 169-50, and *Ueno Zoological Gardens,
Ueno-Kouen, Taitoh-ku, Tokyo 110, Japan
ABSTRACT—Concentrations of testosterone (T) and estradiol-172 (E) in feces were measured in
giant pandas (Ailuropoda melenoleuca). Feces were collected almost monthly from October 1987 to
August 1988 and from June 1989 to July 1990, from captured pandas (a male and a female adult pandas
and their two children) at Ueno Zoo, Tokyo. The elder child born in 1986 was identified later as the
female and the younger one born in 1988 as the male. The concentrations of T in feces of the adult male
was always higher than that in the female when compared in the same month. The concentration of E>
in feces of the adults did not show clear difference between sexes. In the adult female, a high E, peak
was observed in March of 1988, when it showed estrus and was artificially inseminated. No E> peak was
detected in the spring of 1990, when it did not show clear estrus. Concentrations of T and E> in the
children, especially in the younger child, were relatively low and variable. They showed no consistent
seasonal changes.
These results suggest that the sexing of adult pandas is possible by comparing the concentration of T in
feces. Peaks of the fecal steroid content seem to show roughly reproductive condition of an animal,
suggesting a possibility that ovulation can be detected in the female panda by the fecal E> analysis.
© 1992 Zoological Society of Japan
INTRODUCTION
As well-known, the giant panda (Ailuropoda
melenoleuca) is an endangered mammalian species
distributed only in limited areas in China.
Although its ecology has been thoroughly studied
(see Schaller et al. [1]), much remains to be
clarified. Especially, it is important to know
reproductive physiology in wild individuals of this
species. Analysis of sex steroid hormones in blood
plasma is a popular and indispensable method for
studying reproductive physiology of vertebrates.
However, it is impossible or extremely difficult to
collect blood samples from wild individuals of this
species in the field or even from captive individuals
in many cases. In captive individuals, analysis of
urinary steroid can be an alternative, but it is
practically difficult to collect urinary samples espe-
Accepted July 23, 1992
Received June 12, 1992
' Present address:
Minami-dai, Nakano 164, Japan.
Laboratory of Molecular Biology,
Ocean Research Institute, University of Tokyo, 1-15-1,
cially from infant pandas.
Attempts have been made to estimate gonadal
endocrine activity of mammals and birds by ana-
lyzing sex steroids in feces [2-8]. This non-
invasive method was recommended by Risler et al.
[2] as a useful tool in ecophysiological studies and
diagnosis of pregnancy in wild mammals or mam-
mals from which collection of blood or urinary
sample is difficult. Czekala and Lasley [5], and
Stavy et al. [6] employed this method for sexing of
monomorphoic bird species.
Motivated by the study of Risler et al. [2], the
authors intended to study the relation between feal
sex steroids and sex or reproductive condition in
adult and infant giant pandas by measuring sex
steroids in feces which were collected in different
months from two infant giant pandas and their
parents.
1018 K. Kusoxkawa, S. Isuti et al.
MATERIALS AND METHODS
Animals
Two infant giant pandas and their parents reared
in Ueno Zoo, Tokyo were used. The father
(named Fei Fei) was born in 1967 in Sichuan
(China), caputured in 1976 and brought to Ueno
Zoo in November 1982. The mother (Huan Huan)
was born in 1972 in Sichuan (China), captured in
1975 and brought to Ueno Zoo in January 1980.
The infants (Tong Tong and You You) were born
in June 1986 and June 1988, respectively, and have
been reared in Ueno Zoo. It was unable to sex
these infants at the time of their birth morphologi-
cally. Three to four years later, the elder and
younger infants were identified as the female and
the male, respectively, from their behavior and the
difference in the length between the anus and
urogenital opening.
Collection of feces
Fecal samples were collected from the floor of
cages in which animals were kept once a month on
20th day of each month at around 5 am during the
following two periods: the first from October 1987
to August 1988 and the second from June 1989 to
August 1990. The feces were considered to be
excreted between 9 pm and 5 am, since the floor of
the cages used to be cleaned at 9 pm every day.
One of fecal droppings or masses found in the floor
was randomly selected for each animal at each
collection time and frozen in a freezer at —20°C
soon after collection and stored for a few months
until steroid analysis. The mean weight + standard
deviation (SD) of the selected fecal dropping was
56.46 + 20.22 g in the father, 52.44+ 15.52 g in the
mother, 54.39+19.16g in the elder infant and
49.54+22.24 g in the younger infant.
Extraction
We followed the method of Risler er al. [2] with
slight modification. Feces were partially thawed at
the room temperature, and thick threads of bam-
boo contained were removed with forceps. Then,
30 to 100g of the feces were homogenized in a
grinder with 10 volumes of a mixture of ethanol
and acetone (8:2). After adding about 1,000 cpm
of tritiated steroids for determining the recovery
rate, the homogenate was centrifuged at 3,000 rpm
for 10 min at 4°C. The supernatant was filtered
through a cellulose membrane filter (0.2 ~m pore
size, FR-20, Fuji Film Co. Ltd, Tokyo). After
concentrating the volume to about 5 ml by centri-
fuging evaporation in vacuo, the volume was ad-
justed to 20 ml by adding ethanol and water (8:2).
This solution was placed in a freezer at —20°C for
12 to 18 hr to precipitate fat. After centrifuging at
2,000 rpm at —10°C for 20 min, fat was removed.
To the ethanolic supernatant, 5 volumes of pet-
roleum ether was added. After vortexing, the
petroleum ether layer was removed and discarded.
The bottom layer was evaporated to 1 to 2 ml, and
extracted two times with 5 volumes of ethyl ether.
The combined ether phase was washed two times
with 1 ml of 8% sodium bicarbonate solutions with
pH 10 and 8, respectively, and then the ether
phase was evaporated to dryness under nitrogen.
The residue was dissolved in 0.5 ml of methanol
and used as the sample for chromatography.
Chromatography
Estradiol-172 (E>) was separated from testoster-
one and progestrone by the DEAE A-25 column
chromatography according to the method of Risler
et al. [2]. The DEAE A-25 sephadex was changed
from the chloride form to the hydroxide form just
before the column chromatography, and sus-
pended in 100% methanol. Columns were made of
Pasteur pipettes, and packed with 1.5 ml of the
gel. After applying the sample in 0.5 ml methanol,
the neutral steroids were eluted with Sml of
methanol, and then the phenolic steroids with 5 ml
of 0.1 M acetic acid in methanol. Each eluate was
evaporated to dryness and dissolved in 0.5 ml of
methanol.
The mean recovery+SD through the whole
course of the extraction and separation procedures
was 73.6+21.3% in E> and 65.9+17.0% in testos-
terone.
Radioimmunoassay
Testosterone and E> in the chromatographic
fractions were determined by radioimmunoassay
using testosterone-3-(O-carboxymethyl) oximino-
(2-[!"*I]iodo-histamine) of Amersham, England,
Fecal Steroids in Giant Panda 1019
respectively, as radioligands, and the rabbit anti-
testosterone serum (HAC-AA61-02-RBP81) and
the rabbit anti-estradiol serum (HAC-AA63-01-
RBP7), respectively, provided by Prof. Katsumi
Wakabayashi of Gunma University. The separa-
tion of free and bound steroids was performed with
the second antibody method using a goat anti-
rabbit gamma G serum provided by Prof. Katsumi
Wakabayashi. The standard (12.2 pg to 6.25 ng of
testosterone or E>/0.05 ml) or an appropriately
diluted unknown sample (0.05 ml) was preincu-
bated with the corresponding first antiserum (0.05
ml) and buffer (0.1 ml) at 4°C for 24 hr.
Then, the mixture of the sample and antiserum
was reacted with corresponding radioligand
(10,000 cpm/0.05 ml) at 4°C for 48hr. The in-
cubation with the second antiserum (0.1 ml of the
200 times diluted serum) contained with 4%
polyethylene glycol was performed at room
temperature for 3 hr. The mean intra- and inter-
assay coefficients of variation were 1.5 and 4.3%,
respectively, in the testosterone assay. They were
2.9 and 7.87%, respectively, in the E> assay.
Testosterone (ng/g)
1011 1234
87 198
678
8
RESULTS
Testosterone and estradiol concentration in mature
pandas
The testosterone concentration in feces of the
parents varied widely among months, but the
range of the variation was smaller, between 8 and
880 pg/g, in the mother than in the father, be-
tween 105 and 8,910 pg/g (Fig. 1). Especially, the
fecal testosterone level in the mother was signi-
ficantly lower than that in the father when com-
pared in the same month (P<0.01 by the analysis
of variance with two-way layout). In addition,
seasonal changes in the testosterone level were
similar between the two observation periods (one
from June 1987 to August 1988 and the other from
June of 1989 to July of 1990) in both the father and
mother, although data of August and September in
1989 and April and May in 1990 were lacked
accidentally in both individuals.
In the father, there were two peaks of testoster-
one, one in the autumn (October or earlier in 1987
and October in 1989) and the other in the spring or
late winter (April in 1988 and February in 1990).
Corresponding exactly to these spring peaks in the
OQ,
0
5 e)
: 9
Fo)
So :! 3
* (2) 0 12)
ore, A
6 891011121234 67
1989 1990
Fic. 1. Monthly changes of immunoreactive testosterone levels in feces of mature male panda (Fei Fei) (open circles
and dotted line) and mature female panda (Huan Huan) (closed circles and solid line) from June of 1987 to
August of 1988, and from June of 1989 to Jyly of 1990. Samples were collected in 20th of each month. Each point
indicates the mean of duplicate determinations for each fecal dropping.
1020
father, there were peaks of testosterone around
the same time also in the mother. Supporting this,
Kendall’s rank correlation analysis revealed that
there was a statistically significant positive correla-
tion (t=0.75, P<0.05, n=9) in the fecal testo-
sterone concentration between the father and the
mother in the earlier half of the year (from January
to June) but not significant correlation or even
negative correlation (t= —0.60, P>0.05, n=10)
in the variable in the later half of the year (from
July to December).
The concentration of estradiol-178 (E2) in feces
as well as the concentration of testosterone varied
widely among months in both the mother (near 0
to 700 pg/g) and the father (near 0 to 1,064 pg/g)
(Fig. 2). However, unlike the testosterone con-
centration, the estradiol concentration did not
differ clearly between the father and the mother (P
>0.1 by the analysis of variance).
In the father, the E> level elevated conspicuous-
ly in January and February in both observation
periods, showing the highest peaks in February
(Fig. 2). Peaks of E> were also found in June or
1000
800
)
fe)
.
(pg/g
o
fo}
fo}
Estradiol
400
200
67
10 11
1987
Fic. 2.
K. Kusoxkawa, S. Isuui ef al.
Jyly in both observation periods in the father. To
confirm the parallelism of the seasonal changes in
fecal E, between these two periods, the correlation
analysis was applied. There was found a statistical-
ly significant positive correlation (t=0.763, P<
0.05, n=7) in the estradiol concentration in the
father between the two observation periods.
In the mother, the concentration of E> in feces
had the highest peak of the year in November in
both observation periods. High estradiol levels
were also observed in June and July in both
observation periods. Only in the first observation
period, there was an additional high E> peak in
March. The correlation of the estradiol concentra-
tion in the mother between the two observation
periods was not statistically significant (c=0.112,
P>0.05, n=7).
Testosterone and estradiol-178 concentrations in
children
In the elder child, the concentration of testoster-
one in feces was low in the first observation period,
having three peaks in October 1987, January 1988
23 7
1990
8 91011 1
Monthly changes of immunoreactive estradiol levels in feces of mature male panda (Fei Fei) (open circles and
dotted line) and mature female panda (Huan Huan) (closed circles and solid line) from June of 1987 to August of
1988, and from June of 1989 to Jyly of 1990. Samples were collected in 20th of each month. Each point indicates
the mean of duplicate determinations for each fecal dropping.
Fecal Steroids in Giant Panda 1021
ss
Testosterone (ng/g)
7 1011 1234 678 6 891011121 23 67
1987 1988 1989 1990
Fic. 3. Monthly changes of immunoreactive testosterone levels in feces of infant elder panda (Tong Tong) (closed
triangles and solid line) and younger panda (You You) (open triangles and dotted line) from June of 1987 to
August of 1988, and from June of 1989 to July of 1990. Samples were collected in 20th of each month. Each point
indicates the mean of duplicate determinations for each fecal dropping.
=e "
x
800
Estradiol (pg/g)
o
°
ro)
400
7 1011 1234 678 6 7 891011 123 7
1987 1988 1989 1990
Fic. 4. Monthly changes of immunoreactive estradiol levels in feces of infant elder panda (Tong Tong) (closed
triangles and solid line) and younger panda (You You) (open triangles and dotted line) from June of 1987 to
August of 1988, and from June of 1989 to July of 1990. Samples were collected in 20th of each month. Each point
indicates the mean of duplicate determinations for each fecal dropping.
1022
and June 1988, respectively (Fig. 3). However, the
range of the fluctuation was small, and the highest
peak was about 1,000 pg/g. In the second observa-
tion period, the testosterone levels were still low
except the two peaks, one higher than 2,000 pg/g
in December 1989 and the other higher than 4,000
pg/g in February 1990 (Fig. 3). These basal and
peak testosterone levels in the elder child were
clearly lower than the respective levels in the
father. The two peaks in the second observation
period in the elder child were higher than the
highest testosterone peak in the mother, but the
levels in the other months in the elder child were
similar to the level in the mother.
The concentration of E> in feces of the elder
child had three peaks in each of the two observa-
tion periods (Fig. 4). Their locations (October,
January or February and June) coincided approx-
imately between the two observation periods,
although their heights were different.
In the younger child, the concentration of testos-
terone in feces was extremely low, always less than
400 pg/g (Fig.3). The concentrations of E> in
feces of the younger child were low and had three
peaks in November, February and presumably
June (Fig. 4).
DISCUSSION
Validity of the method of fecal sex steroid
analysis must be discussed first. Positive proof of
validity of this method for estrogens including E>
and progesterone was demonstrated first by Adler-
creuz and Jarvenpaa [7] with human feces. They
identified the steroids in the feces by gas chroma-
tography and mass spectrometry. They also found
that 85 to 90% of excreted estrogen occurs in
unconjugated form and also that fecal estrogen
and progesterone well reflect reproductive condi-
tions of men and women. Most or a part of their
results have been confirmed in Macaca by Risler et
al. (2).
In addition, analysis of fecal E> and testosterone
was successfully applied for sexing in three species
of birds by Stavy er al. [6] and for estimation of
gonadal endocrine activity in the Japanese quail
[8], although feces of birds contain urine. Thus,
there will be little doubt about the validity of fecal
K. Kusoxkawa, S. Isuti et al.
sex steroid analysis for assessing gonadal endoc-
rine activity in mammals and birds.
It is obvious that feces of the father contain
higher concentration of testosterone than feces of
the mother. This difference can be due to either
sexual difference or a coincident individual varia-
tion. The former possibility is supported by the
following facts revealed in the present study: 1) the
fecal testosterone showed a peak in the breeding
period of this species, 2) similar seasonal changes
were observed in the two observation periods, 3)
the testosterone levels in the father were higher
than those in the children, and 4) peaks of the
testosterone level appeared simultaneously in the
breeding season also in the mother, although the
peak levels were lower in the mother. It is also
noteworthy that there were additional increases in
fecal testosterone in the autumn in the father. This
fact reminds us the report that the giant panda
breeds some times in the autumn [1]. Furth-
ermore, in Ueno Zoo, artificial collection of sper-
matozoa became possible from October in the
father, and they were available until May or June.
Thus, the possibility of the difference in the testos-
terone concentration in feces between the mother
and the father is obviously high, and hence the
possibility of a coincident individual variation is
low.
We have no proper explanation for the fact that
the father excreted as large amounts of E> in feces
as the mother. However, this is not so surprising,
since Adlercreuz and Jarvenpaa [7] reported that
men and postmenopausal women excreted similar
mounts of estrogen in feces. The presence of the
estradiol peaks in November in both of the two
observation periods in the mother is favorable to
the theory of the presence of the additional breed-
ing period in the autumn. There was a high peak
of estradiol in March of 1988, when the mother
was artificially inseminated and pregnancy was
induced. She gave birth of You You in June of the
same year. However, there was no conspicuous
estradiol peak in March in 1990, when artificial
insemination was attempted again but no pregnan-
cy was induced this time.
Fecal testosterone excreted by the elder child in
the first observation period was as small in amount
as that excreted by the mother. However, in the
Fecal Steroids in Giant Panda
second observation period, there were found two
high peaks of fecal testosterone in December and
February which were higher than the highest peak
in the mother but lower than that in the father.
However, unlike the father, the elder child did not
show high autumnal increase in the fecal testoster-
one. Amounts of excreted testosterone in feces by
the younger child were too low to discuss the
seasonal excretion pattern.
The elder child showed a high fecal estradiol
level in February 1988. This level is as high as the
peak levels observed in the parents. In 1990, there
was found an extremely high estratiol excretion in
feces in the elder child in June. This level was
higher than any of the highest levels observed in
the parents. Interpretation of this estradiol level in
June is difficult.
The younger child excreted small amounts of
estradiol in feces in both 1989 and 1990. These
amounts are similar to those excreted in months of
1987 by the elder child who is 2 years older.
In conclusion, this study suggested with a high
possibility that we can sex adult giant pandas by
analysis of fecal sex steroids, especially testoster-
one, although more examples are needed to con-
clude. It may be also possible to estimate gonadal
endocrine activity of giant pandas indirectly by
analyzing fecal sex steroids successively from the
same individual. However, sexing of infant giant
pandas by analysis of fecal sex steroids may be
difficult.
ACKNOWLEDGMENTS
The authors thank to Mr. T. Hahn, University of
Washington for his reviewing the manuscript and Prof.
1023
K. Wakabayashi for providing the antisera. This work
was supported by grants of the Ueno Zoological graden,
Waseda University and Ministry of Education, Science
and Culture.
REFERENCES
1 Schaller, G. B., Hu, J., Pan, W. and Zhu, J. (1985)
The Giant Pandas of Wolong. The University of
Chicago Press, Chicago.
2 Risler, L., Wasser, S. K. and Sackett, G. P. (1987)
Measurement of excreted steroids in Macaca nemes-
trina. Am. J. Primatol., 12: 91-100.
3 Shille, V. M., Wing, A. E., Lasley, B. L. and Banks,
J. A. (1980) Excretion of radiolabeled estradiol in
the cat (Felis catus, L.): A preliminary report. Zool.
Biol., 3: 201-209.
4 Perez, L. E., Czekala, N. M., Weisenseel, K. A. and
Lasley, B. (1988) Excretion of radiolabeled estradiol
metabolites in the slow loris (Nycticebus coucang).
Am. J. Primatol., 16: 321-330.
5 Czekala, N. M. and Lasley, B. (1977) A technical not
of sex determination in monomorphic birds using
fecal steroid analysis. International Zoo Year book,
17: 209-211.
6 Stavy, M., Gilbert, D. and Martin, R. D. (1979)
Routine determination of sex in monomorphic bird
species using fecal steroid analysis. International
Zool Year book, 19: 209-214.
7 Adilercreuz, H. and Jarvenpaa, P. (1982) Assay of
estrogens in human feces. J. Steroid Biochem., 17:
639-645.
8 Ishii, S. (1985) Estimation of gonadal activity by
means of fecal sex steroid analysis in the Japanese
quail. Report of Japanese Ibis Breeding Project in
1985, Nugata Prefecture, pp. 27-30 (in Japanese).
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ZOOLOGICAL SCIENCE 9: 1025-1035 (1992)
Oviduct 17£-Estradiol Receptor in the Female Lizard, Podarcis s.
sicula, during the Sexual Cycle: Relation to Plasma 17/-
Estradiol Concentration and Its Binding Proteins
M. Paotuccr’, M. M. Di Fiore and G. Crarcia?
Dipartimento di Zoologia, via Mezzocannone, 8. Universita di Napoli, 80134
Napoli, and *Dipartimento di Biologia Evolutiva e Comparata, via
Mezzocannone, 8. Universita di Napoli, 80134 Napoli, Italy
ABSTRACT—In the oviparous lizard, Podarcis s. sicula, 17f-estradiol binding molecules were
characterized in the oviduct and plasma. In addition, their concentration was evaluated throughout the
annual cycle.
In the oviduct, a 17f-estradio] receptor (ER) is present. It shows high affinity for ligand (4.9-7.0 x
10~'°M). It is decreased by ovariectomy, and induced by 17/-estradiol treatment. 17(-estradiol causes
also a ER shift from the cytosol into the nuclei. On sucrose gradient ER behaviour is consistent with the
properties of ER obtained from other 17f-estradiol target organs. At isoelectrofocusing the labeled
© 1992 Zoological Society of Japan
17-estradiol binding molecules fall into three pH ranges: 5.2-5.6, 7.0-7.7, 8,0-8.7.
Nuclear filled ER significantly increases as oviduct grows.
stimulation by 17/-estradiol.
This supports an ER role in oviduct
In the plasma, 17-estradiol binding molecules show many physico-chemical and behavioural
properties of steroid-binding proteins (SBPs).
INTRODUCTION
In the lizard, Podarcis s. sicula, like in most
Oviparous reptiles living in temperate zones, the
female genital apparatus is active during spring
and keeps quiescent for the rest of the year [1]. In
early spring, as animals leave their winter shelters,
several ovarian follicles undergo vitellogenesis,
becoming yolky and ripe in a few weeks. Ovarian
resumption is accompanied by parallel progressive
growth of the oviduct, which rapidly reaches
maturity. Reproductive females usually lay one to
three egg clutches (three to six eggs per clutch at
20-day intervals), starting from the first half of
May. In late spring, the breeding season ceases,
the ovary becomes quiescent, and the oviduct
regresses assuming a typical thread-shaped aspect
(25 Ble
The mature oviduct shows complex gross mor-
Accepted June 6, 1992
Received February 24, 1992
' To whom all correspondence should be addressed.
phology and includes four regions, cranio-
caudally: infundibulum, tuba, uterus and vagina.
Histologically, it is characterized by considerable
hypertrophy of the epithelium and associated
glands. Moreover, oviduct development and activ-
ity give rise to several biochemical events involving
RNA and protein synthesis [4, 5], as well as some
hydrolytic enzyme activity [4]. Lizard eggs are
supplied with little albumen and a coriaceous shell,
which are secreted by tubal and uterine glands,
respectively [6, 2].
Seasonal oviduct growth depends on ovarian sex
hormones, since, in prereproductive females, it
can be easily prevented by ovariectomy and res-
tored by 17f-estradiol or testosterone injections
into spayed females [7]. Moreover the presence of
sex hormone-binding molecules in oviduct cytosol
and nuclei, has been also reported [8].
This work was undertaken with the aim of
defining the properties of oviduct 17/-estradiol
binding molecules in lizards, and ascertaining their
putative receptor nature. Moreover, the concen-
tration of these molecules has been evaluated
1026
throughout the breeding period and in 17f-
estradiol and/or progesterone treated and spayed
females, in relation to the plasma titres of steroid
binding proteins (SBPs) and sex hormones.
MATERIALS AND METHODS
Animals Adult females of Podarcis s. sicula
were caught in the outskirts of Naples during
October 1990-July 1991. Soon after capture, the
animals were anaesthetized with ethyl ether and
bled through a heparinized glass capillary inserted
in the heart. After brief centrifugation, the plasma
was stored in liquid nitrogen until use. At autopsy,
the oviducts were rapidly removed, rinsed in 0.7%
saline solution in order to eliminate any blood
trace, weighed and plunged in liquid nitrogen.
Macroscopical oviduct development and ovarian
follicle diameters were assessed by direct inspec-
tion, and the tissues were divided into four groups,
according to the stage of oviduct development:
1. Quiescent stage (October—February). Ovi-
ducts showed a thread-like aspect (about 19 mg
weight each). The ovary contained only previtel-
logenetic follicles.
2. Recovery stage (March-April). Oviducts
were growing and each of them weighed about 30
mg. In the ovary, several follicles were engaged in
vitellogenetic processes.
3. Full growth stage (April-June). Oviducts
appeared full grown, and each of them weighed
about 80mg. In each ovary 1 to 3 ripe follicles
were ready to ovulate.
4. Secretive stage (June-July). Eggs were con-
tained in the oviduct which weighed about 60 mg.
For experimental studies lizards, caught both in
October and June, were reared in terraria at a
temperature of 28°C, with a photoperiod L:D=
16:8, and fed on meal worms and vegetables ad
libitum. October lizards were ovariectomized;
four weeks later they were divided into four
groups, each of 20 animals, and treated for two
weeks as follows.
Group 1: injected every two days intraperi-
toneally with 0.1 «g of 17P-estradiol (Sigma) dis-
solved in 0.1 ml of 0.7% saline.
Group 2: injected with 0.1 ug of progesterone
(Sigma) dissolved as before.
M. PaoLucci, M. M. Di Fiore AND G. CrarcIA
Group 3: injected with both 0.1 ug of 17f-
estradiol and 0.1 ug of progesterone.
Group 4: injected with 0.1 ml of solvent.
Twenty-four hours after the last injection, the
lizards were killed and processed as reported be-
fore.
June intact lizards with secretive oviducts, were
divided into two groups, each of 10 animals.
Group 1 animals were injected intraperitoneally
with 0.1 ug of 17@-estradiol (Sigma) dissolved in
0.1 ml of 0.7% saline. Group 2 animals received
the solvent alone. The lizards were killed six and
twenty-four hours later, and their oviducts were
utilized as reported above.
Preparation of oviduct subcellular fractions All
procedures were carried out at 0-4°C. Analytical
grade chemicals were used. Oviducts were
weighed, minced and homogenized in 3.5 vol (w/
v) of TEMG (10 mM Tris-HCl, 1 mM EDTA, 1
mM 2-mercaptoethanol, 10% glycerol, pH 7.8),
containing 0.05 M NaCl (homogenization buffer).
The suspension was centrifuged at 800g for 10
min. The supernatant was centrifuged at 105,000
xg for 1 hour in order to obtain the cytosol. The
800g pellet was suspended in 10 vol of buffer
Tris-HCl 10mM containing 3mM MgCh, 2mM
monothioglycerol, 0.25 M sucrose, pH 7.5 (rinsing
buffer), pelletted and rinsed twice with the same
buffer volume. Final pellet was suspended in 3.5
vol. (w/v) of TEMG containing 0.7 M KCI (ex-
traction buffer). This suspension was frozen,
thawed, and left 1 hour in ice bath with occasional
stirring. The suspension was thereafter centri-
fuged at 105,000Xg for 1 hour. The supernatant
constituted the nuclear extract.
Measurement of *H-17{3-estradiol binding in cytosol
and nuclear extract (2,4,6,7-°H)-17-estradiol
(90/110 Ci/mmole) was purchased from Amer-
sham Radiochemical Centre (Amersham, Bucks,
U.K.); unlabeled steroids were obtained from
Sigma Chemical Co. (St. Louis, MO, USA).
Endogenous steroids were previously stripped
from samples by adding an equal volume of
TEMG containing 0.05% (w/v) dextran (Dextran
T-70, Pharmacia Fine Chemicals, Piscataway, NJ
USA) and 0.5% (w/v) active charcoal (Norit A
Estrogen Receptor in Lizard Oviduct
charcoal-Sigma, St. Louis, MO USA). The mix-
ture was vortexed and incubated for 10 min at 4°C.
The charcoal was removed by centrifugation at
3000 rpm for 10min. The steroid-free super-
natants were utilized for all subsequent analyses.
Aliquots (0.2 ml) of cytosol and nuclear extract
were incubated with 5nM labeled 17/-estradiol,
with or without a 100-fold excess of diethylstylbe-
strol (DES) (DES does not bind to steroid binding
proteins in plasma, therefore it is used to discri-
minate between 17/-estradiol binding sites in plas-
ma and in tissues). After 16 hours incubation at
4°C, 0.6ml of active charcoal suspension was
added to the incubation medium. The mixture was
vortexed and kept for 5 min in ice bath; thereafter
it was centrifuged at 800 x g for 10 min at 4°C. The
supernatant was decanted in counting vials and
added with Maxifluor scintillation fluid (Maxifluor,
Packard, Milan. Italy). Radioactivity was mea-
sured in a Liquid Scintillation Spectrometer (Pack-
ard 1600-CA) at 45% counting efficiency.
For Ky determinations, 0.2 ml aliquots of cyto-
sol and nuclear extracts were added to tubes
containing increasing amounts (0.3 to 5nM) of
labeled 17-estradiol, with or without a 100-fold
excess of DES. Incubations were carried out for 16
hours at 4C. Bound and unbound steroids were
separated by adding 0.6 ml of charcoal-dextran
suspension as reported before. Specific binding
data were analyzed according to Scatchard’s
graphic method [9].
Labeled 17f-estradiol unfilled (RU) binding
sites were determined by incubation 0.2 ml of
samples with 5nM labeled 17-estradiol with or
without a 100-fold excess of DES for 30 min at
0°C. Total binding sites (RT) were determined by
incubating similar mixtures for 30 min at 20°C.
The filled binding sites (RF) were calculated by the
formula: RT—RU=RF [10].
For binding specificity evaluation, 0.2 ml sam-
ples were added to 5 nM of labeled 17/-estradiol
with or without increasing amount (10° °-10-° M)
of various unlabeled steroids. Incubation and
separation of bound and unbound steroids were
performed as reported before.
Measurement of *H-17{-estradiol binding in the
plasma Endogenous steroids were removed as
1027
reported above. For Kg determination, plasma
aliquots (0.2 ml), diluted with TEMG to a protein
concentration of 2mg/ml, were used. Proteins
were determined by Lowry et al.’s (1951) method
[11], using BSA as a standard. Samples were
added to tubes containing increasing amounts (0.3
to 20 nM) of labeled 17/-estradiol, with or without
a 100-fold excess of unlabeled 17/-estradiol.
Tubes were incubated at 0°C for 1 hour and there-
after added with 0.6 ml of dextran-charcoal sus-
pension. The mixtures were briefly vortexed and
incubated 1 min at 0°C. After centrifugation,
supernatants were decanted in counting vials and
added with 5 ml Maxifluor scintillation fluid to
evaluate radioactivity as reported before. Binding
specificity was determined by incubating samples
(0.2 ml) with 20 nM of labeled 17-estradiol with
increasing amounts (10° ’-10-° M) of various un-
labeled steroids. Incubation procedures and
radioactivity evaluation were as reported before.
Isoelectrofocusing The method reported by
Matsumada and Goldman was used [12]. Cytosolic
and nuclear extract samples (0.2 ml) were preincu-
bated with 5 nM labeled 17/-estradiol for 1 hour at
4°C; plasma samples were subjected to the same
incubation procedure although with 20 nM labeled
17f-estradiol. A glass column (310.5 cm) was
filled with a mixture of 12.5% sucrose in water
containing 0.01% Triton X-100 and 3% Ampho-
line (Pharmacia, Sweden) pH3.5-10. After a
prerun of 1 hour at 4°C, 200 V, the samples were
layered on top of the gradient. Electrofocusing
was carried out for 16 hours at 4°C, 200 V. At the
end of the run, 0.4 ml aliquots were removed from
the bottom of the column and used for the evalua-
tion of the pH gradient and 17/-estradiol binding
as described above.
Sucrose density gradient Aliquots (0.2 ml) of
cytosol, nuclear extract and plasma were preincu-
bated with labeled 17-estradiol (5 nM for tissue
extracts and 20 nM for plasma) for 1 hour at 4°C.
Afterwards they were layered on the top of 4.4 ml
of 5-20% sucrose linear gradient in TEMG con-
taining 0.7 M KCI, and centrifuged at 189,000 xg
at 4°C for 16 hours. Ovalbumin (3.7 S), albumin
(4.6 S) and catalase (10.5 S) (Sigma) were run as
1028
markers in parallel gradients. After centrifuga-
tion, the gradient were fractionated by collecting
0.2 ml aliquots from the punctured bottom of the
tubes. Samples were reincubated with 5 nM
labeled 17-estradiol with or without a 100-fold
excess of DES or 17f-estradiol, in order to evalu-
ate the specific binding.
Measurement of plasma 17{-estradiol and progeste-
rone A radioimmunoassay (RIA) method
adapted to Podarcis s. sicula plasma was employed
[13]. Sensitivity was 3 pg for both 17-estradiol
(intraassay variability 7%; interassay variability
13%) and progesterone (intraassay variability 6%;
interassay variability 9%).
Statistical analysis Numerical data were analy-
zed by the ANOVA method, followed by the
Duncan multirange test.
RESULTS
Labeled 17/-estradiol binding molecules were
present both in the cytosol and the nuclei of the
lizard oviduct, and showed a high ligand affinity
(Kg=7.0X 107 !°M for cytosol and Kyg=4.9~x
10°-'°M_ for extract) (Fig. 1A, B).
Labeled 17f-estradiol binding molecules were
found also in the plasma, though showing lesser
ligand affinity (Kg=0.8< 10° M) (Fig. 1C). The
affinity values did not undergo significant modifica-
tions in the various phases of the breeding cycle
(Table 1).
Fig. 2 reports the specificity of labeled 17/-
estradiol molecules in cytosol, nuclear extract and
plasma samples. Both 17/-estradiol and DES
competed very well in the nuclear extract, though
specificity curves suggest that labeled 17/-estradiol
molecules bind 17/-estradiol thighter than DES,
while corticosterone, progesterone and testoster-
one competed poorly. 17/-estradiol competed
very well in the cytosol, followed by progesterone,
testosterone, DES and corticosterone. In the
plasma, progesterone, testosterone and corticos-
terone competed with labeled 17/-estradiol mole-
cules, whereas DES was inefficacious.
Table 2 reports the levels of oviduct unfilled
(RU) and filled (RF) labeled 17/-estradiol binding
nuclear
M. PaAotucci, M. M. Di FiorE AND G. CIARCIA
-=10 A
Kd=7.0x10 M
20
2.5 5.0 C
[3H]-Estradiol [dpm x10°]
Kd:0.8x10° M
25 10 20
[SH]-Estradiol [nM]
Fic. 1. ‘Saturation curve and Scatchard analysis of 17-
estradiol binding molecules in the oviduct cytosol
(A) and nuclear extract (B) and in the plasma (C) of
lizard, Podarcis s. sicula during different phases of
the reproductive period. (A) cytosol of quiescent
oviduct; (B) nuclear extract of secretive oviduct; (C)
plasma (February). T=Tolal; S=Specific NS=
Not specific.
sites during the breeding cycle. In the cytosol and
nuclear extract both RU and RF binding sites were
always present; however, RU sites constantly pre-
vailed. RU reached the highest level in quiescent
Estrogen Receptor in Lizard Oviduct 1029
TABLE 1. Kg of the *H-17/-estradiol binding activity in the lizard Podarcis s. sicula oviduct and plasma
during the reproductive cycle
Oviduct stage (n)* Cytosol Nuclear extract Plasma
Quiescent 15 7.0+0.9x 107 '°M 12.1+3.4x10~'°M 0.8+0.3x10-*M
Full-grown 8 n.d.** 1.3+0.2x107'°M 1.5+0.4x10°°M
Secretive 8 8.9+1.1x10-'°M 4.7+0.5x 107 '°M 0.1+0.1x10~-*M
ed
Each value is the mean+SE of three different determinations.
* number of animals for each determination.
** not calculated because abnormal scatchard curue profiles.
100 A Br Cc
% Specific
IO O ONO IOC MOF Mito! 10") io 0? Me’ Pia.
Competitor Concentration (M)
Fic. 2. Labeled 17-estradiol binding specificity in the oviduct cytosol (A) and nuclear extract (B), and in the plasma
(C). Samples refers to Podarcis s. sicula having full grown oviducts. Incubation were carried out with labeled
17f-estradiol and increasing amounts of competitors (10° "-10~° M). Competition is expressed as a percentage
of specific labeled 17f-estradiol binding. (A=Corticosterone; O=Diethylstylbestrol; B@=Testosterone; A=
Progesterone; ®=17/-estradiol).
TABLE 2. Changes in the level of oviduct estradiol binding activity, plasma estradiol binding activity and
plasma estradiol and progesterone in the lizard, Podarcis s. sicula during the reproductive cycle
Oviduct estradiol binding activity (fmol/g tissue) Plasma concentration
Ovduct Plasma estradiol
Mines (n)* Cytosol Nuclear extract binding activity Progesterone Estradiol
8 Receptor Receptor Receptor Receptor (fmol/mg protein) (ng/ml) (ng/ml)
unfilled * filled* unfilled * filled*
Quiescent 6 13108+1543 991+141 47114568 371+35 86.4+ 9.3 ASaelail il, Ase}
Recovery 5 3577+ 365 405+ 49. 32664319 643+65 n.d.** ml," n.d.**
Full-growth 2 220+ 35 145+ 11 2408+223 452+31 145.7+15.1 60.0+9.9 1.4+0.4
Secretive 3 1156+ 131 164+ 13 717+ 68 173+15 71.8+ 6.7 45+1.1 0.2+0.06
Each value is the mean+SE of four different determinations.
* number of animals per each determination.
** not dosed.
# Receptor filled=17/-estradiol binding sites occupied by the endogenous hormone at the time of the assay.
Receptor unfilled=17/-estradiol binding sites not occupied by the endogenous hormone at the time of the assay.
1030 M. Pao.tucci, M. M. Di FiorE AND G. CIARCIA
pH pH pH
B G
8 8 8
6
(3H]- Estradiol [dpm «107 ]
= (e%) on
al ae
1 C
(3H]-Estradiol [dpm «10° ]
ak @ oO
£ (o>)
(3H ]-Estradiol [dpm ~ 10°)
—_ Ww B
b oO
10 1 5 10 1 5 10
Fractions Fractions Fractions
_
a
Fic. 3. Isoelectrofocusing profiles of labeled 17f-estradiol binding molecules in the oviduct cytosol (A) and nuclear
extract (B), and in the plasma (C) of Podarcis s. sicula having full grown oviducts. Specific bound is shown.
Oblique line represents the pH gradient. Each drawing is representative of three different experiments.
' \
2
1
‘
1 5. 10). 15) a20) 525
1 5 10 15 20 25
Fractions Fractions
Fic. 4. Sucrose gradient profile of labeled 17/-estradiol molecules in the nuclear extract (A) and in the plasma (B) of
Podarcis s. sicula having full grown oviducts. Only the specific binding is shown. Each drawing is representative
of three different experiments. The arrow indicates the Albumin (4.6 S) position along the sucrose gradient.
wo
(8H]-Estradiol [dpm «102]
(7H]-Estradiol [dpm « 103]
Estrogen Receptor in Lizard Oviduct
oviducts and decreased as oviduct growth occured
during the breeding period. RF sites showed a
similar behaviour in the cytosol, but they were
significantly higher in nuclear extracts of recover-
ing and full grown oviducts.
The labeled 17/-estradiol binding activity in the
plasma increased during ovarian vitellogenesis; the
same happened for plasma levels of progesterone
and 17-estradiol (Table 2).
At electrofocusing, the labeled 17/-estradiol
binding molecules from both oviduct cytosol and
nuclear extract fell into three pH ranges: 5.2-5.6,
7.0-7.7, and 8.0-8.7 (Fig.3A, B). pH 8.0-8.7
molecules were always present in the oviduct cyto-
sol and nuclear extract, although their level was
lower in recovering and full grown oviducts. pH
7.0-7.7 molecules were found in fairly good
amount in quiescent oviducts, and in the nuclear
extract of secretive oviducts. pH5.2-5.6 mole-
cules were not detected in quiescent oviducts but
were present in the other phases of the oviduct
cycle, except in the cytosol of secretive oviducts.
At electrofocusing, labeled 17/-estradiol bind-
ing proteins in the plasma fell into three pH
ranges: 5.7-5.9, 7.4-7.8, and 8.2-8.7 (Fig. 3C).
These molecules were always detectable, although
pH 5.7-5.9 molecules were the most abundant,
and significantly increased during oviduct growth.
On sucrose gradient, labeled 17/-estradiol bind-
ing molecules of the oviduct cytosol sedimented at
about 4.6S, regardless of the oviduct stage (not
shown). Nuclear extract binding molecules,
however, resolved into two peaks; one peak was
always present and sedimented at about 4.6 S, the
other sedimented at 5.5S and was detectable in
grown and secretive oviducts (Fig. 4A). Plasma
labeled 17f-estradiol binding molecules always
sedimented at about 4.0 S (Fig. 4B).
Figure 5 shows the effects of 17-estradiol injec-
tion on the distribution of labeled 17/-estradiol
binding molecules six hours after hormone admi-
nistration. Estrogen induced decrease of RU and
RF in the cytosol, and of RU in nuclean extract (P
<0.01). Instead, RF in nucleen extract inereased
slightly. 17-estradiol effect was similar twenty-
four hours after the injection (not shown).
Figure 6 reports the effects of ovariectomy and
sex hormone chronic administration on labeled
1031
0.5
—
ER fmol/mg tissue
Oi
a b c d
Cytosol Nuclear
Extract
Fic. 5. Unfilled (GQ) and filled (™) binding sites (ER)
concentration (fmol/mg tissue) in the cytosol and
nuclear extract of Podarcis s. sicula secretive ovi-
duct. a & c=animals injected with solvent; b & d=
animals injected with 17-estradiol. Bars indicate
the S.E.
17-estradiol binding molecules. Ovariectomy was
followed by the RU decrease both in the oviduct
cytosol and nuclear extract (P<0.01), as well as by
the RF increase in the cytosol (P<0.01). In the
oviduct of spayed females injected with 17/-
estradiol or 17f-estradiol plus progesterone, a
further RU and RF decrease occured in the cytosol
(P<0.01), whereas both RU and RF increased in
the nuclear extract (P<0.01). Progesterone admi-
nistration induced a small RU decrease and a RF
increase in the nuclear extract.
DISCUSSION
In the oviparous lizard, Podarcis s. sicula, sea-
sonal oviduct growth and activity depend on ova-
tian steroids, namely 17f-estradiol and testoster-
one [7]. Proteins binding to these hormones have
been found in the oviduct cytosol, and, during the
breeding period, in the nuclei [8]. When injected
in vivo into spayed females, however, 17/-
estradiol is retained in the oviducal tissues, where-
as testosterone is not; therefore the estrogen is
supposed to play a leading role in physiological
oviduct regulation [8]. Our data corroborate this
assumption since we identified an 17/-estradiol
receptor (ER) in the lizard oviduct which behaves
according to the oviduct annual cycle.
178-estradiol binding molecules bind the ligand
with high affinity (Kg=7.010~'° M for the cyto-
1032
38 A
34
e
=
(o)
Estradiol Receptor fmol/mg tissu
oO
1
a
M. Pao.ucct, M. M. Di FiorE AND G. CIARCIA
B
15
10
5
b c d e a b c d e
Fic. 6. Effects of ovariectomy and 17f-estradiol and/or progesterone treatment on unfilled (G) and filled (m)
17f-estradiol binding sites concentration in the oviduct cytosol (A) and nuclear extract (B) of Podarcis s. sicula
captured in October. a=intact lizards; b=ovariectomized lizards treated with solvent (saline); c=ovariecto-
mized lizards treated with 17f-estradiol; d=ovariectomized lizards treated with 17-estradiol plus progesterone;
e=ovariectomized lizards treated with progesterone. Bars indicate the S.E.
sol and 4.9x10~-'°M for the nuclear extract).
Ligand affinity does not significantly change during
the cycle. These parameters are consistent with
the properties of the oviduct 17@-estradiol receptor
(ER) of several lower vertebrate species: elasmob-
ranchs [14]; reptiles [15]; birds [16-19], and of the
mammalian oviduct and uterus [20].
However, in the cytosol of full-grown oviduct,
the specific 17-estradiol binding resolves in an
abnormal profile of the Scatchard plot (not
shown), which does not allow Kg calculation. The
meaning of this result is obscure.
The hormone-binding sites are present in the
cytosol and nuclei of oviduct tissues. Binding
activity is decreased by ovariectomy and is res-
tored in spayed females after 17(-estradiol treat-
ment. .In spayed 17f-estradiol treated females,
moreover, cytosolic unfilled and filled binding sites
decrease whereas nuclear filled sites increase. In
oviducts of female treated with 17-estradiol for a
short time, a displacement of the binding sites
from the cytosol to the nucleus occurs, although
the total amount appears to be decreased, as
reported in several other studies [20, 21].
In our opinion this behaviour rules out an arti-
factual origin of cytosol binding sites as shown in
several vertebrate systems [22, 23]. Quite a similar
model has been reported for liver ER of the
Estrogen Receptor in Lizard Oviduct
salmon [24] and Rana esculenta [21].
The level of 17f-estradiol binding sites changes
during the oviduct cycle. In the cytosol, it is higher
in quiescent oviducts, but decreases as the organ
grows. During growth a significant increase of
nuclear filled sites occurs, which is coupled with an
increase in plasma 17/-estradiol and progesterone
titres as well as plasma levels of steroid binding
proteins (SBPs).
In Podarcis s. sicula oviducts, the increase in
17f-estradiol-induced binding sites is not counter-
acted by progesterone administration, as reported
for avian and mammalian systems [25-27]. This
result, however, is in line with that obtained in the
oviduct of the turtle Trachemys scripta [28], and
proposes a difference in 17f-estradiol receptor
regulation between higher vertebrates and lizards.
On sucrose gradient 17-estradiol binding mole-
cules resolve into two discrete peaks with sedi-
mentation coefficients of 4.6S and5.5S. The 4.6
S peak is always present, whereas the 5.5 S peak
has been found only in the nuclear extract of
growing or secretive oviducts, and, therefore,
might represent an activated form of the 17/-
estradiol receptor. Changes in the ER sedimenta-
tion coefficient following activation have been
reported [29-31]. The sedimentation coefficient
values of lizard oviduct putative ER are consistent
with those found for ER from several vertebrate
systems, sedimented at high ionic strength [32].
At electrofocusing, the oviduct 17/-estradiol
binding molecules fall into three discrete pH
ranges, id. 5.2-5.6, 7.0-7.7, and 8.0-8.7. It is
difficult to interpret these results, although the
absence of 5.2-5.6 molecules in quiescent oviducts
and their appearance in the nuclear compartment
of growing and secretive oviducts suggest that they
might represent the activated receptor. A change
in receptor pI following activation has been found
in the androgen receptor of the rat prostate [33].
In Podarcis s. sicula plasma, 17-estradiol bind-
ing proteins (SBPs) behave as those found in the
plasma of other lower vertebrates [34-36],
although in Podarcis s. sicula, binding is displaced
also by corticosterone, a property found only in
plasma SBP of Nerodia sipedon [37]. The sedi-
mentation coefficient of lizard SBP is 4.0 S, a value
similar to that reported for SBP of Alligator missis-
1033
sipiensis [35], but lower than that found in SBP of
other vertebrates [38-41].
At electrofocusing, lizard SBP fall into three pH
ranges, i.d. 5.7-5.9, 7.4-7.8 and 8.2-8.7, although
the first form is the most abundant. The plasma
level of pH 5.7—5.9 molecules changes during the
oviduct cycle; it is higher when vitellogenesis prog-
resses in the ovary, and its level is related to
17f-estradiol and progesterone plasma titres. This
behaviour confirms its role as plasma sex-hormone
carrier [42-44]. SBP changes related to the sexual
cycle phase have been reported in the females of
Taricha granulosa [45], Alligator mississipiensis
[35] and in the male of Podarcis s. sicula [36].
ACKNOWLEDGMENTS
This work was supported by grants of the Ministero
della Pubblica Istruzione, Italy (40% and 60%).
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Riley, D., Kleis-San Francisco, S. M. and Callard, I.
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ZOOLOGICAL SCIENCE 9: 1037-1045 (1992)
© 1992 Zoological Society of Japan
Endogenous Testosterone Controls Humoral Immunity
in the Lizard, Chalcides ocellatus
ABDEL HAKIM SAAD, MOHAMED HESHAM MANSOUR,
MANSOUR EL YAZJI and NASsrRi BADIR
Zoology Department, Faculty of Science, Cairo University,
Cairo 12613, Egypt
ABSTRACT— Study of the correlation between endogenous blood levels of testosterone (TS) and the
humoral immune response of male lizards, Chalcides ocellatus led to three major findings: 1) Long-term
testosterone propionate (TP) treatment in “summer” induced an elevation of serum TS and complete
abrogation of primary anti-rat erythrocytes (RRBC) antibody response similar to normal intact lizards
during “spring”; 2) Bilateral orchidectomy of male lizards during “spring” showed increased response to
RRBC and suggested that orchidectomy potentiated immunity and more particularly definite stimula-
tion of humoral immunity; 3) Long-term TP treatment of orchidectomized lizards failed to suppress
immune response to RRBC. Indeed, orchidectomized lizards given TP had response to RRBC similar
to male intact controls. The results are discussed from the perspective of the role played by TS in
mediating the seasonal rhythms that affect reptilian immunity.
INTRODUCTION
Many decades ago scientists and clinicans alike
observed that there were striking differences be-
tween the immune responsiveness of males and
that of females [1]. In general, female had super-
ior humoral and cell-mediated immunity [2, 3]. It
is very likely that this difference in the immune
response between the sexes is mediated by the
action of sex steroids hormones on the im-
munoreactive cell populations [4]. Since the major
source of these sex steroids is the gonads, it follows
that removal of these organs by gonadectomy
might be expected to alter the immune response.
Although gonadectomy has been shown to alter
the cell-mediated immune response, there is a
considerable controversy concerning the effects of
gonadectomy on antibody production [5-9]. In
contrast to the wealth of information obtained
from study of gonadectomy and immunocompe-
tence of mammalian vertebrates, data available
about fish [10] and amphibians [11] are inadequate
and as yet limited. At the reptilian level, no
Accepted June 18, 1992
Received February 14, 1992.
attempts were made to study the influence of
gonadectomy on different immunological para-
meters.
The present study is mainly based on previous
observations from our laboratory showing that the
immune system of the lizard, Chalcides ocellatus
was moderately active during April throughout
June, reaching its maximum development during
July/August [12, 13]. This slowly vernal building
of immune response was ascribed to be due to high
levels of endogenous testosterone (TS) [12].
Therefore, the present study was initiated to ex-
amine the effect of adult orchidectomy and testos-
terone treatment on the immune response of C.
ocellatus. his study was conducted in view of
contributing further information to the endoge-
nous hormonal factors that mediate seasonal
changes in immunity of reptiles.
MATERIALS AND METHODS
Lizards
Sexually mature male lizards, Chalcides ocella-
tus (Scincidae, non-hibernator, viviparous), 30—40
g in weight were collected from gradens, fields and
1038
arid regions in the vicinity of Cairo. Lizards were
maintained as described previously [12] and given
wood lice and tap water ad libitum. The study was
performed from March through September
(temperature ranges of 25-38°C).
Preparation of cell suspensions
Lymphoid cells were separated from lizard
spleen exactly as described in detail earlier [13].
After three washes in cold RPMI 1640 medium
(Sigma Chemicals Co., St. Louis, MO., USA),
lymphocytes were counted using the trypan blue
dye exclusion test.
Blood testosterone (TS) determination
Serum testosterone was measured following
essentially the method described earlier [12] using
a commercial radio-immunoassay kit (Coat-A-
Count, Diagnostic Products Corporation, CA,
USA). |*I-labelled TS was supplied as the
radioactive tracer. Tetosterone showed less than
1.5% cross reactivity with corticosterone, a major
serum corticosteroids in C. ocellatus [13]. The
mean intra- and interassay coefficient of variation
of assays fell within the range of 4.0-7.0% and
8.0-15.0%, respectively. In these assays, the
minimum detectable dose on the standard curve
was 10 pg. Recovery of labelled and unlabelled
steroids standard was 90-93%. Values shown in
the “Results” are corrected for recovery.
Primary immunization with RRBC
Animals were allowed to acclimate to ambient
environmental conditions for few days before im-
munization. The lizards received intraperitoneal
(i.p.) immunizing injections of 0.5 ml of 10% rat
erythrocytes (RRBC) suspension in phosphate-
buffered saline (PBS), pH=7.2. Unimmunized
(control) lizards were injected i.p. with 0.5 ml
PBS, pH=7.2 and included in every such experi-
ments. After sacrifice by decapitation, blood was
collected and allowed to clot at room temperature.
Serum was then heat-inactivated at 56°C for 30
min. and stored for use in the haemagglutination
test.
Rosette-forming cell (RFC) assay
Groups of animals were sacrificed at a specified
A. H. Saap, M. H. Mansour et al.
intervals after immunization. A single cell suspen-
sion from the spleen was prepared as described
and the viability was determined by trypan blue
exclusion method. The rosette-forming cell (RFC)
assay was followed to assess the number of anti-
gen-binding cells as described earlier [14]. On the
basis of actual number of cells for each sample, the
RFC per 10° spleen cells was computed for each
spleen.
Plaque-forming cell (PFC) assay
The technique of Cunningham and Szenberg
[15] as modified for lizards by Kanakambika and
Muthukkaruppan [16] was followed to assess the
number of haemolytic antibody-producing cells.
Plaques were counted under low power with the
aid of a dark field phase contrast microscope. The
number of PFC/10° viable spleen cells was calcu-
lated.
Determination of antibody (Ab)
Haemagglutination (HA) titrations of the sera
were performed in microtiter plates using two-fold
serial dilution as described in detail previously
[12]. Titers were expressed as the logs of the
highest dilution showing microscopic agglutina-
tion.
Bilateral orchidectomy
Adult male lizards were allowed to acclimate to
laboratory conditions for at least one week before
operation. Surgical operations were performed
under ether anaethesia. Operation sites were
washed with distilled water followed by 70% ethyl
alcohol. Skin was firstly cut by a sharp cissors,
while the abdominal muscles cut by means of a
small scalpel. The left then the right testes were
separated from their blood supply by means of a
cautery then cut off their respective vasa deferen-
tia. Sulpha and neomycin was sprayed on the
wound then sewed by means of a small curved
surgical needle. Operated animals were placed in
a clean glass terrarium for one day to avoid sepsis.
In corresponding sham-operated controls, the
testes were delivered to the wound surface, re-
turned to the abdominal cavity and the wound
closed. No death occurred in either operated or
sham-operated groups during the course of the
Testosterone and Immunity in Lizards
experiment. Both were kept at ambient tempera-
ture under normal conditions in the laboratory and
given live wood lice and water ad libitum.
Testosterone propinate (TP) treatment
Intact, orchidectomized
lizards were allowed to acclimate to ambient en-
vironemtnal conditions a few days before treat-
ment. All lizards received at 5 day-intervals a total
of 5 i.p. injections of 50 #g testosterone propion-
ate (Sigma)/g body weight/injection. Another
group of lizards was injected at the same intervals
with 0.3 ml of PBS, pH =7.2 and used as control.
No death occurred in either TP-treated or control
groups during the course of the experiment. Both
were kept at ambient temperature under normal
conditions in the laboratory and given wood lice
and water ad libitum.
sham-operated or
Statistical analysis
Student’s f-test was used to determine levels of
significance between control and experimental
groups. Differences were considered to be signi-
ficant at P values <0.05.
RESULTS
Effect of long-term TP. treatment on intact lizards:
Circulating TS levels:
As illustrated in Figure 1, TS levels of intact
males injected with PBS exhibited minor changes
within the normal range of about 9.5-26.3 ng/ml.
However, TS levels after multiple injections of TP
were markedly elevated to the range of 18-140 ng/
ml, and stabilized at this level until the end of the
experiment. Ten days after cessation of TP, a
decline to basal values proceeded sharply.
Primary anti-RRBC response:
In mid-summer, male lizards were given five 1.p.
injections of TP (50 “g/g body weight/injection; 5
days apart). Another group of male lizards re-
ceived PBS and served as controls. Two days later,
all TP-treated and PBS-injected lizards received an
i.p. injection of RRBC suspension. RFC and PFC
assays were performed on individual animals using
spleen cell suspensions on different days after
1039
Testosterone level (ng/ml)
0 5 10 15 20 30 40
= a & = =
Days after TP treatment
Fic. 1. The levels of serum TS in intact lizards, C.
ocellatus, administered with multiple injections of 50
pg, TP/g body weight/injection. During mid-
summer, lizards received 5 injections, 5 days-apart,
of TP suspension. Each point depicts the level of TS
of serum sample pooled each from two animals.
Day O represents TS levels of intact lizards injected
with PBS.
immunization. Titrations of serum Ab titers were
carried out simultaneously.
As depicted in Figure2A, TP-treated male
lizards elicited a low number of RFC comparable
to that enumerated in PBS-injected lizards.
However, significant (P<0.01) differences could
be observed at the peak day (Fig. 2A). As shown
in Fig. 2B, although the number of PFC higher in
PBS-injected males than in TP-treated lizards, no
significant differences could be observed at the
peak day. Indeed, the shape of the curves was
otherwise quite similar. As depicted in Figure 2C,
in PBS-injected lizards, low levels of Ab titer was
detected below background level on day 7. There-
fore, the titer rose quickly exhibiting a peak at day
21 and remained elevated until day 28. However,
in TP-treated lizards, Ab titer increased steadily
reaching its peak value at day 21, then gradually
declined.
Effect of bilateral orchidectomy on intact lizards:
Circulating TS level:
Adult male lizards, C. ocellatus were collected
1040 A. H. Saap, M. H. Mansour et al.
during early spring and some of these lizards were
orchidectomized during mid-March. Another
group was sham-operated and used as controls.
Figure 3 illustrates the serum TS levels in male
o
i—}
aT
leen cells
~
o
agg Nes
2” aa sham- operated and orchidectomized lizards dur-
is ing March through July. Sham- operated lizards
sea exhibited high levels of TS being in the range of
i 110-140 ng/ml. However, bilateral orchidectomy
0 in C. ocellatus lowered the level of TS to about 2—
18 ng/ml. These abrogated levels were sustained
2 and lasted long, so that the mean values for all
2 operated lizards, at any given point were signi-
ficantly (P<0.001) different from sham-operated
controls.
3 7 14 21 28
Days post immunization
a
o
a
o
6
Number of PFC /10 spleen cells
>
o
w
o
Testosterone level (ng/ml)
iy
o
10
sham- 15 day 1 2 3 month
operated
0 iL Time after orchidectomy |
fa : P HH The levels of serum TS obtained from adult C
DAYS POS! imewametiion Fic. 3. e levels of serum obtained from adult C.
ocellatus after bilateral orchidectomy. Each point
on the curve represents the level of TS obtained
from an individual sample pooled from two animals.
Day O represents TS levels of sham-operated
ai @9) lizards.
Fic. 2. Kinetics of RFC response (A), PFC response
(B) and mean serum haemagglutinin titers (C) of
normal lizards, C. ocellatus, administered with mul-
tiple injections of 50 ng TP/g body weight/injec-
tion. During mid-summer, lizards received 5 injec-
tions, 5 days-apart, of TP suspension. Two days
later, lizards were received 1.p. injection of 0.5 ml of
10% RRBC suspension. Each point represent the
mean response of 4-6 separate animals and the
Reciprocal log, HA titer
vertical bars indicate standard error of the mean.
@—e Normal intact lizards, @---@ Normal intact
lizards injected with TP. *=0.05<P<0.01 and NS
7 14 21 28 SS
a Days post immunization =not significant.
Testosterone and Immunity in Lizards 1041
Primary anti-RRBC response:
Groups of adult lizards were sham-operated or
orchidectomized during the second week of
March. Two separated experiments were per-
formed: the first after one month and the second
after three months of operation. During each
experiment, orchidectomized and sham-operated
lizards were received a single i.p. injection of
RRBC suspension on day (0. RFC and PFC assays
were carried out using spleen cell suspension on
different days after immunization. Titration of
serum Ab levels were carried out simultaneously.
As depicted in Figure 4A, in sham-operated
lizards, the number of RFC increased from back-
ground levels to a peak level on day 14 and then
dropped sharply between day 21 and day 28. In
contrast, both groups of orchidectomized lizards
receiving RRBC suspension manifested a signi-
ficant (P<0.01) increase in RFC on day 7. The
number of RFC remained elevated on day 14, then
gradually declined. As shown in Figure 4B, admi-
nistration of RRBC suspension elicited a peak
response of PFC on day 14, then sharply declined.
However, both groups of orchidectomized lizards
showed a sparp (P>0.01) increase in the number
of PFC which was peaked on day 14 and then
gradually decreased (Fig. 4B). When sham-
operated lizards received RRBC suspension, low
levels of Ab titer was detectable below background
level on day 7 (Fig. 4C). Thereafter, the titer rose
slowly exhibited a peak at day 21, then gradually
declined at day 28. However, in both groups of
orchidectomized lizards, a sharp increase in cricu-
lating Ab titer occurred in day 7. Thereafter, a
gradual increase occurred in the Ab titer (Fig. 4C).
Fic. 4. Kinetics of RFC response (A), PFC response
(B) and mean haemagglutinin titers (C) of C. ocella-
tus after bilateral orchidectomy. Animals were
immumized i.p. on day 0 with 0.5 ml of 10% RRBC
suspension. Each point represents the mean re-
sponse of 4-6 separate animals and the vertical bars
indicate standard error of the mean. *=0.05< P<
0.01 and NS=not significant. @—@ sham-operated
lizards; A—A orchidectomized lizards, one month
after operation; ©—Q© orchidectomized lizards,
three months after operation. *=0.05<P<0.01
and NS=not significant.
3
Number of RFCx10 /10 spleen cells
el n hh
ie 3 7 4 21 28
Days post immunization
Number of PFC /10° spleen cells
HA titer
Reciprocal log
SS
7] con 21 28
&
Days post immunization
1042
Effect of long-term TP-treatment on orchidecto-
mized lizards:
Circulating TS levels:
TP was administered to orchidectomized lizards
to determine whether increasing the concentration
of serum TS would result in the modulation of
immune reactivity. Those lizards were orchidecto-
mized during May. One month later, one group of
orchidectomized lizards received five fractionated
dose of TP (50 ug/g body weight/injection; 5
days-apart). Control orchidectomized lizards re-
ceived five i.p. injections of PBS.
As illustrated Figure5, TS levels of
orchidectomized lizards injected with PBS exhi-
in
= 120
E
oD e
Cc
= tbo} e e
ee e
9 2 e e
® 80+ ‘3
e
o
5 a
() e
a7) e
e)
Ef
2
"|
e
at 5 10 15 20 30 40
rN PS os = ~~
Days after TP treatment
Fic. 5. The levels of serum TS in orchidectomized C.
ocellatus administered with multiple injections of 50
yg, testosterone propionate (TP)/g body weight/
injection. One month after orchidectomy, lizards
received 5 injections, 5 days-apart, of TP. Each
point depicts the level of TS of serum sample pooled
each from two animals. Day O represents TS levels
of orchidectomized lizards injected with PBS.
Fic. 6. Kinetics of RFC response (A), PFC response
(B) and mean serum haemagglutinin titers (C) of
orchidectomized C. ocellatus administered with mul-
tiple injections of 50 ug TP/g body weight/injec-
tion. One month after orchidectomy, lizards re-
ceived 5 injections, 5 days-apart, of TP suspension.
Two days after injection, lizards were received i.p.
injection of 0.5 ml of 10% RRBC suspension.
orchidectomized lizards; orchidecto-
mized lizards injected with TP. *=0.05<P<0.01
and NS=not significant.
6
3
RFC x10 /10 spleen cells
Number of
3 7 14 21
a
6
Number of PFC /10 spleen cells
titer
log HA
9,
Reciprocal
ao
>
w
NR
—
A. H. SAAD, M. H. Mansour et al.
28
Days post immunization
a 3 7 14 21 28
Days post immunization
7 14 21 28
> Days post immunization
Testosterone and Immunity in Lizards
bited minor changes within the normal range of
1.1-5.7 ng/ml. In comparison, orchidectomized
lizards receiving five injections of TP showed a
sharp increase in serum TS levels to about 45-120
ng/ml which maintained until day 20.
Primary anti-RRBC response:
TP was administered to orchidectomized lizards
to determine whether increasing TS levels would
result in modulation of the immune response.
Lizards were orchidectomized during May. One
month later, one group of orchidectomized lizards
received i.p. five fractionated dose of TP (50 ug/g
body weight/injection; 5 days-apart). Control
orchidectomized lizards received i.p. five injec-
tions of PBS. Two days after the last injection,
lizards received i.p. injection of RRBC suspen-
sion.
As shown in Figure6A, in TP-treated
orchidectomized lizards, the kinetics of RFC was
similar to that demonstrated in orchidectomized
control lizards. Moreover, no significant differ-
ences were essentially observed between the two
animal groups at the peak day. As shown in Figure
6B, in both PBS-injected and TP-treated
orchidectomized lizards, the shape of the curves
was Otherwise quite similar. Although both groups
of lizards showed a high response, no significant
differences were observed at the peak day. As
depicted in Figure 6C, despite the fact that in
TP-treated orchidectomized lizards, the peak of
primary Ab response to RRBC was somewhat
delayed and diminished in comparison with PBS-
injected orchidectomized lizards, statistical differ-
ences in humoral response at some time point were
essentially not significant.
DISCUSSION
In the present study experimental administra-
tion of exogenous TP in lizards produced changes
in serum TS levels which closely mimicked those
Occurring as part of response of lizards to the
seasonal environment [12]. As expected, long-
term TP-treatment of intact summer lizards led to
a complete and an irreversible abrogation of their
proliferative response to RRBC. The results indi-
cated that the cell(s) responsible for these func-
1043
tional alterations in lizards were particularly TP-
sensitive; the target cell(s), however, remained
undefined. It is obvious that several doses of
exogenous TP induced a high and more long
standing elevation of TS resulting in a depressed
immune reactivity, which simulates the natural
situation lizards experienced annually in spring
[12].
In view of the above, the authors hypothesized
that lowering an animal’s blood TS levels by
“orchidectomy” did alter the immune response
and that alterations might be opposite to those
induced by the administration of exogenous TP.
Thus, bilateral orchidectomy was performed to
adult male C. ocellatus in early spring in order to
suppress the high TS levels at that time of the year
[12].
lizards based on actually reproducing endocrine
The immune reconstitution of “spring”
environment is theoretically possible but ex-
perimentally difficult maneuver. Yet, the outcome
has been fruitful. Although bilateral orchidectomy
strongly reduced serum TS levels in C. ocellatus, it
did not result in its complete disappearance.
However, these concentrations were similar to the
low values previously recorded throughout sum-
mer months [12]. Moreover, orchidectomized
lizards showed increased response to RRBC and
the rate of lymphoid tissue hyperplasia correlated
with this increased reactivity (data not shown).
These data, therefore, suggested that
orchidectomy potentiated immunity in general and
more particularly humoral one. Whether this
effect is due to the differentiation of lymphoid
precursor cells to immunocompetent cells and/or
the proliferation of pre-existing competent cells,
which might be present in low level in the other
lymphoid organs is difficult to decide.
Although TS deprivation showed to cause im-
munological potentiation, the central point re-
maining is whether the effects are due directly to
withdrawal of TS or they result from interference
with the hormonal milieu. Therefore, TP was
administered to orchidectomized lizards to deter-
mine whether increasing the concentration of
blood TS would result in modulation of the im-
mune reactivity. One month after orchidectomy,
orchidectomized lizards received five fractionated
doses of TP. These orchidectomized lizards exhi-
1044
bited a spring-like sustained rise of serum TS levels
until the end of the experiment. However, phys-
iologic doses of TP used in the current study failed
to suppress immune responses to RRBC in
orchidectomized lizards. In fact, orchidectomized
lizards given TP had response to RRBC which
were similar to male These
findings suggested that the presence of TS caused
immune function to be “normal” in orchidecto-
mized lizards.
To our knowledge, no such study has been
available in reptiles. However, the scientific litera-
ture dealing with sex hormones effect on verte-
brate immune responses appeared to be confused
and scant. While in marine teleost, Sebasticus
marmaratus, the humoral reactivity of mature
females to RRBC was lower than males or imma-
ture females in the spawning season, yet exoge-
nous administration of sex hormones or
orchidectomy did not display influence on Ab
production during the mating season [10]. The
thymus and spleen were morphometrically analy-
zed in orchidectomized frogs, Rana perezi [11].
However, the authors failed to interpret their data,
since such important physiological processes were
undobtly the direct effect of TS deprivation [11].
In addition, recent studies indicated that neonatal
gonadectomy in chicken exerted two effects on the
humoral immune response: suppressive in young
birds [17] and stimulatory in older chicken [18].
Moreover, orchidectomy resulted in immuno-
intact controls.
potentiation in many mammalian systems. For
example, orchidectomy
against viral or fungal [5-7] and bacterial infec-
tions [8] and parasitic infections [9]. Moreover,
orchidectomized animals reject allografts rapidly
[19] and accelerated graft-versus-host reactions
[20].
lymphoblasts transformation in culture [21] and
potentiated humoral immune response to several
However, in some ex-
perimental situations, orchidectomy had no effect
on the immune system [8].
In conclusion, all these results confirm the im-
increased protection
Furthermore, gonadectomy augmented T-
heteroantigens [22, 23].
portance of sex hormones as causative agents of
the seasonal variations of reptilian immune system
and emphasized the lymphocyte destructtion as the
main effect of sex hormones on the immune system
A. H. Saap, M. H. MAnsour et al.
of lower vertebrates. Mechanisms of action of sex
hormones, target cells for sex hormones in the
lymphoid organs and the way of interaction be-
tween sex hormones and the immune axis in lower
vertebrates deserve further research.
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Testosterone and Immunity in Lizards
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ZOOLOGICAL SCIENCE 9: 1047-1053 (1992)
Effects of Photoperiod, Pinealectomy and Ophthalmectomy
on Circulating Melatonin Rhythms in the
Goldfish, Carassius auratus
Hiroaki Kezuka!, MasayukI I1Go*, KtvosHt FURUKAWA,
Katsumi Arpa® and Isao HANyu
Department of Fisheries, Faculty of Agriculture, The University
of Tokyo, Bunkyo, Tokyo 113, Japan
ABSTRACT—Effects of photoperiod, pinealectomy and ophthalmectomy on circulating melatonin
rhythms were studied in the goldfish, Carassius auratus. Under light-dark (LD) 16:8 or LD 8:16
photoperiod, plasma melatonin levels exhibited diurnal rhythms with high titers during the scotophase
and low titers during the photophase. When the fish were transferred from LD 12:12 to continuous
dark conditions, plasma melatonin levels exhibited circadian changes during the first 3 days under
continuous dark-ness (DD). The rhythms, however, became indistrinct during days 7-8 and 14-15.
When the fish were transferred to continuous light conditions, plasma melatonin remained at low levels.
Pinealectomy abolished high melatonin levels in the plasma at mid-dark, but ophthalmectomy did not.
These results clearly indicate that circulating melatonin levels are photoperiod-dependent and shows
circadian rhythms under DD conditions, and that the plasma melatonin rhythm is mainly generated by
© 1992 Zoological Society of Japan
the pineal gland.
INTRODUCTION
The pineal gland of vertebrates synthesizes and
secretes its indole hormone, melatonin (N-acetyl-
5-methoxytryptamine) into the blood and cere-
brospinal flud. Melatonin has been considered as
the time-keeping hormone because of its cyclic
appearance: melatonin levels in the pineal gland,
blood, and cerebrospinal fluid fluctuate in a rhyth-
mic fashion that is coincident with a given photo-
period. Under light-dark (LD) cycles, high meato-
nin titers were observed during the scotophase
while low values were seen during the photophase
in all vertebrate classes including fishes [1-13].
Accepted June 25, 1992
Received January 16, 1992
Present address: Advanced Research Laboratory,
Hitachi, Ltd., Hatoyama-machi, Saitama 350-03,
Japan.
> Present address: Department of Anatomy, St.
Marianna University School of Medicine, Miyamae-
ku, Kawasaki 216, Japan.
To whom reprint requests should be addressed.
The pineal gland of fishes is considered to be a
circadian oscillator, since behavioral analysis re-
vealed that the pineal gland is implicated in the
control of circadian organization and rhythmicity
in locomotor activities [14, 15]. In addition, en-
dogenous rhythmicity in melatonin secretion from
the pineal gland under continuous dark (DD)
conditions in vitro has been reported in three
teleost species, the pike (Esox lucius) [16], the
gold-fish (Carassius auratus) [17, 18], and the
white sucker (Catostomus commersoni) [19].
These results suggest that melatonin secreted from
the pineal gland is playing an important role as an
internal zeitgeber in controlling bioligcal rhythms
in fishes.
Photoperiod is considered to be the most impor-
tant environmental factor which modulates mela-
tonin rhythms. Reports, however, on the effects of
long or short photoperiod on circulating melatonin
thythms in fishes were restricted to the rainbow
trout Oncorhynchus mykiss [20] and the common
carp Cyprinus carpio [12], and no report concern-
ing endogenous rhythms of melatonin in vivo
1048
under continuous dark (DD) or continuous light
(LL) conditions is available.
Melatonin and the enzymes responsible for
melatonin biosynthesis (i.e. serotonin N-
acetyltransferase and hydroxyindole-O-
methyltransferase) have been reported to localize
in the retina (for review, see [21]). These results
indicate that melatonin is synthesized not only in
the pineal gland but also in the retina. Pinealec-
tomy and ophthalmectomy experiments revealed
that contributions of the pineal gland and the
retina to circulating melatonin rhythms exhibited
inter-species differences. In the Japanese quail,
for example, the retina contributes to the blood
level of melatonin and daily cycles of circulating
melatonin in pinealectomized animals [22]. In the
chicken, on the other hand, pinealectomy abo-
lished circulating melatonin rhythms [23]. In case
of teleost fish, melatonin synthesis in the pineal
gland and the retina have been reported in several
species [11, 13, 16-19, 24-26], but how these two
tissues contribute to the circulating melatonin level
is not fully understood.
The present study was conducted to examine the
effects of photoperiod on circulating melatonin
rhythms, and to confirm the main source of circu-
lating melatonin in the goldfish.
MATERIALS AND METHODS
Experimental _ fishes old goldfish
(Carassius auratus) were purchased from a local
dealer. They were reared in indoor stock tanks at
24°C under natural photoperiod at The Fisheries
Laboratory, The University of Tokyo (Maisaka,
Shizuoka, Japan) until used. Fish were fed com-
mercial trout pellets ad libitum.
Two-year
Experiment 1 Ninety male goldfish weighing
50-150 g were used in this experiment. In Octo-
ber, 45 fish were transferred into each of 2 indoor
experimental tanks, and acclimated under LD
16:8 (lights on 0400-2000 hr) or LD 8:16 (lights
on 0800-1600 hr) at 24°C for 2 weeks. Blood
samples were taken at 2—4 hr intervals (n=5) for
twenty-four hr.
Experiment 2 Three hundred goldfish weighing
H. Kezuka, M. Iico et al.
38-174 g were used in this experiment. In July,
150 fish were transferred into each of two indoor
experimental tanks, and acclimated under LD
12:12 (lights on 0600-1800 hr) at 24°C for 2
weeks. Blood samples were taken at 1200 hr on
Day 0 (57-97 g females, n=7; 53-110 g males, n=
5) and at 0400 hr on day 1 (47-112 g females, n=6;
47-100 g males, n=5) to examine sexual differ-
ences in plasma melatonin levels. The light condi-
tions were changed to DD or LL conditions from
0600 hr on day 1. Blood samples were taken every
4 hr (n=5) at 0800 hr on day 1 to 0800 hr on day 3
(days 1-3), at 1200 hr on day 7 to 1200 hr on day 8
(days 7-8), and at 1200 hr on day 14 to 1200 hr on
day 15 (days 14-15).
Experiment 3 Forty-two glodfish weighing 72-
154g were used in this experiment. After the
acclimation under LD 12:12 (lights on 0600-1800
hr) at 24°C for 3 weeks in August, they were
pinealectomized (PINX, n=9), sham-
pinealectomized (Sham, n=8), ophthalmecto-
mized (EYEX, n=8), or pinealectomized and
ophthalmectomized (PINX+EYEX, n=9).
Pinealectomy and sham-pinealectomy were
accomplished according to the method of De
Vlaming [27] and ophthalmectomy was performed
following the procedure of Fenwick [28]. Animals
were identified by fin-clips. Intact control (IN-
TACT, n=6) were only fin-clipped. One week
after operation, blood samples were taken at mid-
dark (2300-0100 hr) and mid-light (1100-1300 hr,
36 hr after the sampling for middark).
Sample collection and RIA Blood samples
were taken according to the procedure by Kezuka
et al. .{12] under with 0.06% 2-
phenoxyethanol in Exp. | or with 0.05% ethyl-p-
aminobenzoate in Exps. 2 and 3. The anesthesia
did not interfere with the RIA. Blood samples
were centrifuged at 3000 rpm for 20 minutes, and
anesthesia
plasma was stored at —20°C until the assay.
Melatonin levels in the plasma were measured
by the RIA after a partial purification with Sep-
pak Cys cartridge as previously described and
validated for the goldfish plasma [12]. The mini-
mum detectable level of the RIA was 32 pg/ml
plasma in these experiments.
Plasma Melatonin Rhythms in Goldfish
Statistics The difference of means was analyzed
by ANOVA and Duncan’s multiple range test, or
by paired (test.
RESULTS
Circulating melatonin rhythms under long or short
daylength
Plasma melatonin levels exhibited distinct daily
rhythms under both long (LD 16:8) and short LD
8:16) daylength as shown in Figure 1. The lowest
levels were seen at 1900 hr under LD 16:8 and at
1500 hr under LD 8:16. Levels were observed to
increase after lights-off, and remained elevated
during the scotophase. The highest levels were
1200
LD 16:8 hy
900
600 # ie
3004
12 16 19210 35 8 12
LD 8:16
MELATONIN (pg/ml)
b
(=)
(=)
12 1517 20 O 4 79 12
CLOCK TIME (hr)
Fic. 1. Circulating melatonin rhythms under long (LD
16:8, upper) or short (LD 8:16, lower) photo-
period. Each point represents the mean+SE (n=
5). Solid bars and open bars along the X-axis
indicate the scotophase and the photophase, respec-
tively. Significance: under LD 16:8, +, compared
with the values during the photophase (1200, 1600
and 1900 hr on Day 1 and at 0500, 0800 and 1200 hr
on Day 2); under LD 8:16, +, compared with the
values at 1500 hr on Day 1 and at 0900 and 1200 hr
on Day 2; *, compared with the values at 1200 and
1700 hr on Day 1. Levels of significance: one
symbol, P<0.05; two symbols, P<0.01.
1049
observed at 0000 hr under LD 16:8 and at 0400 hr
under LD 8:16. Subsequently, melatonin levels
started to decrease, although the fish were still in
the scotophase, and returned to the basal levels
after lights were turned on.
Under LD 16:8, the values during the scotoph-
ase (2100 hr on Day 1 and 0000 and 0300 hr on Day
2) were significantly higher than those during the
photophase (1200, 1600 and 1900 hr on Day 1 and
0500, 0800 and 1200hr on Day 2) (P<0.01).
Under LD 8:16, the values at 2000 hr on Day 1
and 0000 and 0400 hr on Day 2 were significantly
higher than those at 1500 hr on Day 1 and 0900 and
1200 hr on Day 2 (P<0.01), and the values at 0000
and 0400 hr on Day 2 were significantly higher
than those at 1200 and 1700 hr on Day 1 (P<0.01).
The value at 0700 hr on Day 2 was significantly
higher than those at 1500 hr on Day 1 and 0900 and
1200 hr on Day 2 (P<0.05).
Sexual difference in circulating melatonin levels
Plasma melatonin levels in females and males
were 154+18 and 154+38 pg/ml at 1200 hr on
Day 0, and 460 +48 and 667 +121 pg/ml at 0400 hr
on Day 1 (mean+SE), respectively. There was no
detectable difference in plasma melatonin levels
between sexes either during the photophase (at
1200 hr on Day 0) or during the scotophase (at
0400 hr on Day 1). Therefore, female and male
fish were not separately dealt with in the following
experiments.
Circulating melatonin rhythms under DD or LL
conditions
Changes in plasma melatonin levels under DD
or LL conditions are shown in Figure 2. Under the
LD cycles used for the acclimation, melatonin
levels exhibited day-night fluctuations: The values
during the scotophase (0400 hr on Day 1) were
significantly higher than those during the photoph-
ase (1200 hr on Day 0, P<0.01). When the fish
were transferred into DD conditions, plasma mela-
tonin levels exhibited circadian changes during the
first 3 days with high levels during the subjective
scotophase and low during the subjective photoph-
ase. The level at 0400 hr on Day 2 was significantly
higher than tose at 1200 hr on Day 1 and those at
1200 on Day 2 (P<0.05), and the levels at 2000 hr
1050
1200
900
600
300
MELATONIN (pg/ml)
12, 0 12 0 12 =O
DAY O DAY 1
H. Kezuka, M. Ico et al.
ACCLIMATORY
PHOTOPERIOD
12 0 1212 0 12
DAY 2 DAY 3 DAYS7-8 DAYS 14-15
CLOCK TIME (hr)
Fic. 2.
Circulating melatonin rhythms under DD (@) or LL (™) conditions. Each point represents the mean+SE
(1200 hr on Day 0, n=12; 0400 hr on Day 1, n=11; under DD or LL, n=S each). Solid bars and open bars along
the X-axis represent the scotophase and the photophase of the acclimatory photoperiod, respectively. Signi-
ficance: *, compared with the value at 1200 hr on Day 1; +, compared with the value at 1200 hr on Day 1; A,
compared with the value at 1200 hr on Day 2. Levels of significance: one symbol, P<0.05; two symbols, P<0.01.
on Day 2 and at 0000 and 0400 hr on Day 3 were
significantly higher than those at 1200 hr on Day 2
(P<0.05). During Days 7-8, circadian-like
changes in plasma melatonin levels disappeared.
During Days 14-15, melatonin levels remained at
elevated levels. In contrast, under LL conditions,
plams melatonin levels remained at low titers
throughout the experiment.
Effects of PINX and/or EYEX on circulating
melatonin rhythms
Plasma melatonin levels at mid-dark and mid-
light one week after the operation are shown in
Figure 3. At mid-dark, melatonin levels in the
PINX and PINX+EYEX fish were significantly
lower than those in the Intact, Sham, and EYEX
groups (P<0.01). No significant difference was
observed among mid-light values of all groups.
In the Intact, Sham, and EYEX groups, melato-
nin levels at mid-dark were significantly higher
than those at mid-light (P<0.01). There was no
significant difference between values at mid-dark
and mid-light in the PINX, whereas in the PINX +
EYEX the value at mid-dark was significantly
lower than those at mid-day (P<0.01).
600
(J INTACT
(ll SHAM
= EYEX
3 400 fe PINX
= PINX+EYEX
2
e
=
w 200
=
0
MID-DARK MID-LIGHT
Fic. 3. Plasma melatonin levels at mid-dark and mid-
light in the INTACT (n=6), SHAM (n=8), EYEX
(n=8), PINX (n=9), and PINX+EYEX fish (n=
9). Each point represents the mean+SE. _ Signi-
ficance: *, compared with the value in Sham at
mid-dark; s+, compared with the value at mid-light in
each group. Levels of significance: two symbols, P
<0.01.
DISCUSSION
In the goldfish, plasma melatonin levels exhi-
bited clear daily rhythms both under long and
short daylength; low during the photophase and
Plasma Melatonin Rhythms in Goldfish 1051
high during the scotophase with peak near mid-
dark phase. Our results were basically the same as
those reported in other vertebrate species [1-13].
These results indicate that daily fluctuations of
melatonin in the body fluid are a common phe-
nomenon in vertebrates and that photoperiod is
one of the most important factors controlling mela-
tonin rhythms.
Under DD conditions, plasma melatonin levels
exhibited endogenous rhythms during Days 1-3;
high melatonin levels were observed during the
subjective scotophase and low titers were seen
during the subjective photophase. This suggests
that circulating melatonin rhythms are driven by a
circadian oscillator. These rhythms disappeared
during Days 7-8 and 14-15. Several explanations
can be presented for this observation such as: large
individual variations in free-running period, damp-
ing of oscillation, or desynchronization of oscilla-
tors under DD conditions [29]. In contrast, under
LL conditions, plasma melatonin concentrations
remained at low levels and failed to exhibit daily
rhythms. This indicates that exposure to light has a
strong inhibitory effect on melatonin secretion.
Similar results were previously observed in
organ culture experiments of the goldfish pineal
gland in vitro [17, 18]: under LD cycles, the pineal
gland secreted melatonin during the scotophase;
circadian rhythms of melatonin secretion were
observed under DD conditions; and melatonin
secretion is suppressed under LL conditions. The
coincidence of these in vivo and in vitro results
support the idea that melatonin secreted from the
pineal gland contributes to the blood melatonin
rhythms.
Because melatonin synthesis has also been re-
ported in the retina of some vertebrates including
fishes in addition to the pineal gland [5, 8, 13, 21-
25, 30-33], we examined effects of pinealectomy
and/or ophthalmectomy on circulating melatonin
levels to confirm the main source of circulating
melatonin in the goldfish. In the PINX and PINX
+EYEX groups, high values at mid-dark dis-
appeared, whereas in the EYEX group significant-
ly higher values were maintained at mid-dark.
These results clearly indicate that the pineal gland,
and not the retina, is the main organ which sec-
retes melatonin into the circulatory system in this
species. We cannot, however, exclude the possi-
bility that a small amount of melatonin is secreted
from the retina. Although the difference is not
significant, plasma melatonin levels at mid-dark
were lower in the PINX+EYEX group that those
in the PINX group. In addition, we recently found
in the goldfish that melatonin contents in the eye
also show daily fluctuations (ligo and Aida, in
preparation).
Interestingly, plasma melatonin in the goldfish
was still at detectable level after pinealectomy and
ophthalmectomy, and the plasma melatonin levels
at mid-light was significantly higher than those at
mid-dark in PINX+EYEX fish. This residual
melatonin may be simply due to secretion from
remnants of the pineal tissue after pinealectomy,
and/or secretion from other parts of the body.
The Harderian gland and the intestine have been
suggested at the extrapineal-extraretinal source of
melatonin in mammals and birds [31-33].
The pineal gland plays an important role as the
photoneuroendocrine transducer in vertebrates,
and melatonin secreted from the pineal gland into
the blood is thought to serve as an internal zeitge-
ber. In seasonally breeding mammals, melatonin
mediates photoperiodic information and the mod-
ulation of melatonin secretory profiles is involved
in the determination of the reproductive season
(for review, see [34]). In temperate-zone fishes,
photoperiod is one of the most important factors
determining the spawning season [35-37]. Photo-
period controls daily melatonin cycles in teleost
fishes [10-13, 20] as well as in mammals [1-3],
however, the relationship between seasonal repro-
duction and melatonin rhythms is still unknown.
Further investigations will reveal the exact role of
the pineal gland and circulating melatonin rhythms
in teleost fish.
ACKNOWLEDGMENTS
We express our thanks to Dr. C. S. Tamaru, The
Oceanic Institute, Hawaii, for reading the manuscript.
This study was supported in part by a grant-in-aid (Bio
Media Program) from the Ministry of Agriculture, Fore-
stry, and Fisheries.
9
10
1052
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De Vlaming, V. L. (1975) Effects of pinealectomy
on gonadal activity in the cyprinid teleost, Notemi-
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Fenwick, J. C. (1970) Effects of pinealectomy and
bilateral enucleation on the phototactic response
and on the conditioned response to the light of the
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Takahashi, J. S.. Hamm, H. and Menaker, M.
(1980) Circadian rhythms of melatonin release from
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Hattori, A., Yamamoto, K., Ohtera, K., Hara, M.,
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Gern, W. A., Duvall, D. and Nervina, J. M. (1986)
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89-94.
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ZOOLOGICAL SCIENCE 9: 1055-1060 (1992)
© 1992 Zoological Society of Japan
Intermediary Metabolism in Castrated/Thyroidectomized
Calotes versicolor: Regulation by Thyroxine
and Testosterone
VALSA S. PETER and OOMMEN V. OOMMEN
Department of Zoology, University of Kerala, Kariavattom,
Trivandrum 695 581, India
ABSTRACT—Thyroidectomy did not change blood glucose level and acid phosphatase (Ac. Pase; liver
and kidney) activity, and castration reduced the same in Calotes versicolor, when compared to their
respective sham-operated controls. Castration or thyroidectomy decreased the activities of glucose-6-
phosphatase (G-6-Pase; liver and kidney), glutamic oxaloacetic transaminase (GOT; liver and heart),
glutamic pyruvic transaminase (GPT; liver) and urea (liver and kidney) concentration. Thyroidectomy
markedly elevated hepatic cholesterol level and castration did not change its concentration. Adminis-
tration of thyroxine (T4) to castrated and testosterone (T) to thyroidectomized animals stimulated the
activities of G-6-Pase, Ac. Pase, GOT, GPT, the concentrations of glucose and urea, and decreased
liver cholesterol concentration.
It is suggested that both T, and T are effective in stimulating
intermediary metabolism in lizards, irrespective of the removal of either thyroid or gonad.
INTRODUCTION
The relation between the activity of thyroid
gland and reproduction has been reported in cer-
tain species of reptiles [1, 2]. Testosterone restores
the decrease in oxygen consumption of isolated
tissues, following thyroidectomy, in the lizard Ca-
lotes versicolor and the snake, Natrix piscator [3].
Histological studies revealed that gonadal regres-
sion after thyroidectomy and thyroid hyperplasia
after gonadectomy are regained to normal by
replacement therapy of thyroxine to thyroidecto-
mized and testosterone propionate to gonadecto-
mized C. versicolor [4]. Administration of differ-
ent doses of thyroxine to thyroidectomized lizards
[5] and testosterone to castrated animals [6] pro-
vide evidence that these hormones play an impor-
tant role in intermediary metabolism of C. versico-
lor. However, little is known about the thyroid-
gonad interrelationship on reptilian intermediary
metabolism. In this paper, we report the effects of
administration of testosterone to thyroidectomized
and thyroxine to gonadectomized lizards on the
Accepted June 29, 1992
Received December 12, 1990
biochemical constituents and the enzymes associ-
ated with intermediary metabolism in different
tissues of the lizard, C. versicolor.
MATERIALS AND METHODS
Adult male Calotes of 24+4 g body wt captured
from the local areas of Trivandrum during
December/January were housed in metallic wire-
netted cages. The animals were provided with
minced beef liver and fresh water given ad libitum
and were fasted for two days prior to sacrifice.
After a week of acclimation to the laboratory
conditions (28+2°C and 12L:12D), the animals
from each of the two sets were divided into four
groups of five animals each. Group one animals in
both first and second sets served as sham-operated,
hormone vehicle (propylene glycol/alkaline
saline) injected controls. Animals in groups II, III
and IV in the first set were thyroidectomized as
described elsewhere [5] and groups 2, 3 and 4 in
the second set were castrated under open ether
anaesthesia. A small incision was made on the left
dorsolateral side of the animal, just above the
pelvic girdle, and the two testes were removed in
toto in asceptic conditions. While group II animals
1056
served as thyroidectomized controls, groups II and
IV were administered with 25 and 50 ug of testos-
terone (T) respectively. Animals of group 2 served
as castrated controls and groups 3 and 4 were
administered with 5 and 10 ug of thyroxine (T4),
respectively.
Hormone administration was started five days
after operation, intramuscularly, on alternate legs,
between 7.00 and 7.30 A.M. The daily dose of
hormone administered was in 0.1 ml of the respec-
tive vehicle and treatments were for a period of
five days. L-thyroxine (Sigma Chemical Co.) was
dissolved in alkaline saline (pH 9.0), and testoster-
one (Sigma Chemical Co.) was dissolved in prop-
ylene glycol (BDH, India). Twenty-four hr after
the last injection, blood was drawn from the heart
of anaesthetised animals which were then sac-
rificed by decapitation. Liver, kidney and heart
were removed, chilled and a 10% homogenate was
prepared in 0.25 M sucrose medium. After sub-
jecting the homogenate to differential centrifuga-
tion (10,000 g) for 10min at 4°C, the post-
mitochondrial supernatant was collected.
The specific activities of glucose-6-phosphatase
(G-6-Pase) [7, 8], acid phosphatase (Ac. Pase) [9],
glutamic pyruvic transaminase (GPT; also called
alanine aminotransferase) and glutamic oxaloace-
tic transaminase (GOT; also called aspartic amino-
transferase) [10] and the estimation of the bioche-
mical constituents, glucose, [11], urea [12] and
cholesterol [13] were colorimetrically done. The
details of assay procedure are described elsewhere
[S].
Data collected from five animals in each group
were analysed by one-way classification of ANO-
VA followed by multiple range test [14, 15].
RESULTS
Thyroidectomy did not change the blood glucose
level (Fig. 1), and castration reduced the same
(Fig. 2), when compared to respective sham-
operated lizards. Castration and thyroidectomy
decreased the activities of G-6-Pase (liver and
kidney), Ac. Pase, (liver and kidney), GOT (liver
and heart), GPT (liver) and urea concentration
(liver and kidney), except that of Ac. Pase, which
remained unaltered in thyroidectomized speci-
V. S. PETER AND O. V. OOMMEN
mens when compared to their respective sham-
operated lizards (Figs.1, 2). Thyroidectomy
markedly elevated hepatic cholesterol level (Fig.
1), and castration had no effect (Fig. 2).
Injections of 25 or 50 ug of T in thyroidecto-
mized lizards stimulated the activities of G-6-Pase,
Ac. Pase, GOT, GPT, and the concentrations of
glucose and urea; markedly decreased hepatic
cholesterol when compared to thyroidectomized
lizards (Fig. 1). The lower dose of T (25 yg) did
not stimulate Ac. Pase activity (Fig. 1).
Administration of 5 or 10 ug of T4 to castrated
lizards significantly increased the glycemic level
and urea concentration when compared to cas-
trates (Fig. 2). The activity of G-6-Pase was signi-
ficantly stimulated by the administration of 5 and
10 ug of Ty whereas only 10 4g dose stimulated the
activities of Ac. Pase, GOT and GPT (Fig. 2). Ten
vg Ts had a hypocholesterolemic effect, while 5 ug
dose had none in castrated lizards (Fig. 2).
DISCUSSION
Earlier studies have shown that thyroid and
testes have well known seasonal variations in their
activity in C. versicolor [4, 16]. The hyperglycemia
appeared after Ty, administration to castrates may
be due to the action of exogenous Ty. The
hyperglycemia after T, or T administration in
castrated or thyroidectomized lizards indicates that
in the absence of T, T, may increase blood glucose
level and vice versa. This suggests an interrela-
tionship of thyroid and gonad probably mediated
through hypothalamic-pituitary axis. The main
effects of thyroid hormones on glycemic level seem
to be hyperglycemic [17]. Recently, Jacob and
Oommen [5] have reported that thyroidectomy
does not change the glucose level, and castration
produces a hypoglycemic effect in C. versicolor [6].
Josekumar and Oommen [4] have suggested in C.
versicolor that gonadectomy results in hyperplasia
of the thyroid follicle which is characterized by
high output of thyrotropin (TSH) due to low level
of Ty in blood. They have also shown that
thyroidectomy in C. versicolor results in degenera-
tion of testis, the degree of which varies in diffe-
rent periods of the year [4]. In reptiles, thyroidec-
tomy has been reported to have a direct effect on
Thyroid Gonad Relationship in Calotes 1057
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Fic. 1. Effect of administration of testosterone (T) on certain enzyme activities and biochemical constituents in
thyroidectomized (Tx) Calotes versicolor. Each column is mean+S.D. of five animals. Statistics according to
multiple range test. SO: Sham-operated. *P<0.05 **P<0.01.
1058
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400
40
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270
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Fic. 2.
SO
V. S. PETER AND O. V. OOMMEN
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4 J
Effect of administration of thyroxine (Ty) on certain enzyme activities and biochemical constituents in
castrated (C) Calotes versicolor. Each column is mean+SD of five animals. Statistics according to multiple range
test. SO: Sham-operated CC: castrated control. *P<0.05 **P<0.01.
Thyroid Gonad Relationship in Calotes
Leydig cells index which is highly sensitive to
gonadotropins [18].
Hyperglycemia after Ty or T administration may
be due to increased rate of gluconeogenesis as
G-6-Pase is related to homeostatic regulation of
blood glucose. Decreased activity of this enzyme
after thyroidectomy or castration gives evidence
for the reduced rate of gluconeogenesis. It has
been shown that Ty injection to thyroidectomized
and T to castrated lizards produce an increased
G-6-Pase and Ac. Pase activities [5-7]. Van Rees
et al. [19] have reported that castration decreases
both pituitary and serum TSH levels whereas
testosterone propionate increases TSH levels in
castrated male rats.
Ac. Pase, a lysosomal enzyme which hydrolyses
phosphate esters in an acid environment is associ-
ated with synthetic activities depending upon the
physiological state of the tissues involved in the
process. The increased activity of Ac. Pase by T or
T, in thyroidectomized and castrated lizards re-
spectively, is an indication of increased lysosomal
activities in liver and kidney. A significant stimula-
tion in the activities of the two transaminases,
GOT and GPT after injections of T, to castrated
and T to thyroidectomized animals may suggest
decreased proteolysis, as these transaminases are
concerned with amino acid catabolism and gluco-
neogenesis [20]. It is also likely that in the absence
or presence of T,/T either one hormone or both
may participate in increasing transamination and
gluconeogenesis.
The increased concentration of urea, following
administration of T4 and T to castrated and thy-
roidectomized lizards respectively, illustrates that
both hormones are equally effective in increasing
urea concentration in this high energy demanding
poikilotherm. The decreased cholesterol concen-
tration after T, and T administration to castrated
and thyroidectomized animals reveals the lipolytic
action of these hormones in lizards. Thus it seems
that both T, and T promote lipid breakdown,
facilitating availability of substrates for oxidation
in this metabolically active animal. Stimulation of
oxidative metabolism by thyroid and gonadal hor-
mones has been reported in reptiles by Oommen
and Sreedeviamma [21].
The present investigation suggests the biochemi-
1059
cal influence of testosterone in the absence of
thyroid function and thyroxine in the absence of
testicular function. The study helps to confirm the
previous suggestion [23] that poikilotherms de-
pend on androgens to a considerable extent for the
maintenance of intermediary metabolism and also
confirms the earlier reported histological effects of
thyroid ablation on gonadal function and vice versa
[4]. It seems that thyroid and testis in C. versicolor
exhibit a direct interrelationship which may in-
volve gonadotropins and thyrotropins. The in-
volvement of hypothalamo-hypophysial axis in the
modulation of thyroid and gonadal function has
been known in vertebrates. The hypophysial gly-
coprotein hormones, TSH, FSH and LH in some
cases overlap their biological activities. It has been
proposed that thyroid function evolved parallel
with endocrine control of reproduction and that
the basic function for thyroid hormones is associ-
ated primitively with maturation of gonads [22].
Thyroxine exerts a negative feed back on both
TSH producing cells and gonadotropic cells; con-
versely, gonadal hormones influence both these
pituitary cells [24]. The control over TSH and
gonadotrops remain closely linked and there are
common pathways to the control of both these cell
types in brain of teleost [25]. Further, in teleost
fishes the control of TSH is evolved from the
control of original gonadotropins which indicate a
close relationship between thyroid function and
reproduction. Thyrotropin release has not been
examined but is presumed to be under hypothala-
mic stimulatory control in reptiles [27]. However,
synthetic mammalian TRH does not stimulate the
thyroid gland of the turtle [28]. It was also
reported that changes in thyroid activity influence
the output of gonadotropins in birds [26]. Such a
parallel relationship between the function of gonad
and thyroid is suggested in Calotes versicolor also.
ACKNOWLEDGMENTS
The authors are grateful to the Head, Department of
Zoology, for providing laboratory facilities and one of
the authors (VSP) thanks CSIR, New Delhi for granting
a fellowship.
10
13
1060
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Menon, J., Hiradhar, P. K., Paul, V. F. and Shah,
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Fiske, C. H. and Subbarow, Y. (1925) The col-
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Nigam, V. N., Davidson, H. M. and Fishman, W.
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Nelson, J. (1944) A photometric adaptation of the
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79-100.
Abell, L. L., Levy, B. B., Brodie, B. B. and
Kendall, F. E. (1952) A simplified method for the
estimation of total cholesterol in serum and demon-
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V. S. PETER AND O. V. OOMMEN
stration of its specificity. J. Biol. Chem., 195: 357-
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tistical Methods”. Oxford & IBH Publishing Co.,
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variation in thyroid hormonogenesis in the Indian
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451-462.
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(1983) Plasma thyroid hormones in cyclostomes: Do
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Van Rees, G. P., Noach, E. L. and Van Dieten, J.
A. M. J. (1965) Influence of testosterone on the
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thyroid and testicular hormones in the oxidative
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gy”, Lea & Febiger, Philadelphia, 2nd ed. p. 89.
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Thyrotropin-releasing hormone and thyrotropin in
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Hil.
ZOOLOGICAL SCIENCE 9: 1061-1066 (1992)
Immunocytochemical Detection of Prolactin and Growth Hormone
Cells in the Pituitary during Early Development
of the Japanese Eel, Anguilla japonica
Emi ARAKAWA, ToyoJI KANEKO, KATSUMI TSUKAMOTO
and TETsuyA HIRANO
Ocean Research Institute, University of Tokyo
Nakano, Tokyo 164, Japan
ABSTRACT—The occurrence and development of prolactin (PRL) and growth hormone (GH) cells
were investigated in the Japanese eel by means of an immunocytochemical method. Both PRL and GH
cells were detected in the pituitary in all the specimens of the leptocephali, even in the smallest (10.0
mm in total length). In the leptocephalus, the mean percentage of PRL-cell area to the whole pituitary
area (% PRL) was 6.5+1.2%, the % GH being 15.5+2.0%. Both % PRL and % GH exhibited a
tendency to decrease as leptocephali grew larger. In the glass eel, caught just before upstream
migration, the % PRL was 12.6+0.5%, almost twice as much as that in the leptocephalus, whereas the
% GH was 16.7+1.3%, similar to the value in the leptocephalus. The activation of PRL cells in the
glass eel suggests that PRL has an osmoregulatory role in freshwater adaptation during the upstream
migration. Our findings also suggest that GH is important for larval growth and, possibly, osmoregula-
© 1992 Zoological Society of Japan
tion in seawater during their early life stages.
INTRODUCTION
Prolactin (RPL) is well known as an important
hormone for freshwater adaptation in many eury-
haline species [1, 2]. Activation of PRL cells or
increase in plasma PRL has been observed after
transfer from seawater to fresh water. On the
other hand, there is a large body of evidence that
growth hormone (GH) is involved in seawater
adaptation especially in salmonids. Increase in
plasma GH after transfer from fresh water to
seawater has been reported in several salmonid
species [3-9].
The eels belong to the catadromous fishes; they
experience both seawater and fresh water during
their life-long migration. Ecological studies have
shown that they spawn eggs offshore and leaf-
shaped larvae (leptocephali) drift in the current
toward the coasts. After metamorphosis, juveniles
(glass eels) migrate upstream and stay in the river
Accepted July 20, 1992
Received June 20, 1992
or the lake for 5-10 years; they grow in fresh water
and return to the ocean for spawning. However,
early developmental stages of the Japanese eel,
Anguilla japonica, have hardly been studied, be-
cause of very limited availability of eggs and
leptocephali. Furthermore, the spawning area of
the Japanese eel had been unknown until quite
recently.
In June—Jyly 1991, the expedition of the Haku-
ho-Maru, a research vessel of the Ocean Research
Institute, University of Tokyo, succeeded in sam-
pling more than 900 pre-leptocephali and lep-
tocephali, and determined the spawning area of
the Japanese eel to be in the North Equatorial
Current west of the Mariana Islands [10]. The
pre-leptocephali and leptocephali captured ranged
from 7.9 to 34.2 mm in total length, much smaller
and thus younger than those that had ever been
collected.
In order to clarify the development of
osmoregulatory mechanism during the migration
of the Japanese eel, we examined the occurrence
and development of both PRL and GH cells by
1062
means of immunocytochemistry, using valuable
samples of the preleptocephali and leptocephali,
together with glass eels just before upsteam migra-
tion to the river as well as sexually immature,
cultured eels. This is the first report on the
identification of PRL and GH cells during the early
developmental stages of the Japanese eel.
MATERIALS AND METHODS
Pre-leptocephali and leptocephali of the
Japanese eel (10.0-30.0 mm in total length), esti-
mated to be 12-48 days old, were collected in the
area west of Mariana Islands (salinity: 34.5%c) in
June-July 1991 [10]. In the present paper, the
term leptocephalus designates both pre-
leptocephalus and leptocephalus for convenience.
Glass eels of the same species (55-60 mm) were
caught on the coast of Taiwan (salinity: 30%c) in
November 1991. Sexually immature, cultured
eels, weighing about 200 g, were obtained from a
commercial dealer in Tokyo.
Head portions of leptocephali and glass eels and
pituitaries of cultured eels were fixed for 24h in
Bouin’s solution and preserved in 70% ethanol.
Later, they were embedded in paraplast, and
sagittal sections were cut serially at 4 um thick-
ness. To identify PRL and GH cells in the
pituitary, the sections were stained immunocy-
tochemically according to the ABC method [11]
using commercial reagents (Vectastain ABC Kit,
Vector Laboratories). Briefly, the sections were
sequentially incubated with (1) 0.6% hydrogen
peroxide (H,O3>) for 30 min to inactivate endoge-
nous peroxidase activity, (2) 2% normal goat
serum for 30 min to reduce non-specific binding,
(3) the specific antisera against eel PRL [12] and
eel GH [13], overnight at 4°C, (4) biotinylated
anti-rabbit IgG for 30min, (5) avidin-biotin-
peroxidase complex (ABC) for 1 h, and (6) 0.02%
3,3’-diaminobenzidine tetrahydrochloride contain-
ing 0.005% HO, for 4-5 min.
To determine the optimal dilution of the anti-
sera, preliminary experiments were conducted,
where the pituitary of the cultured eel was im-
munocytochemically stained with serial dilutions
of the antisera against PRL and GH. The best
staining was obtained at a dilution of 1: 16000 in
E. ARAKAWA, T.
KANEKO et al.
both PRL and GH cells. In the case of PRL cells,
however, a lower dilution of 1:8000 was also
employed, since the RPL cells in the leptocephalus
were stained rather faintly at the dilution of 1:
16000. The specificity of the immunoreaction was
confirmed by preabsorbing the antisera with re-
spective antigens.
For the quantitative analysis, percentages of
PRL- and GH-cell areas to the whole pituitary
area (% PRL and % GH) were estimated as
follows: Serial sections at intervals of 16-20 um in
leptocephali and glass eels and about 100 «m in
cultured eels were immunocytochemically stained
with either anti-PRL or anti-GH sera. The areas
of PRL and GH cells and the whole pituitary were
measured on microphotographs with a tablet digi-
tizer. The % PRL and % GH were calculated as
percentages of the total PRL- and GH-cell areas,
respectively, to the total pituitary area. Significant
differences were determined by Student’s ftest or
Cochran-Cox test after F-test comparison of
variance.
RESULTS
In the leptocephalus, the pituitary appeared as a
cell cluster or mass, located beneath the hypotha-
lamus, and was barely distinguishable as a defini-
tive organ (Fig. 1A, B). Both PRL and GH cells
were detectable in all specimens of leptocephali
(10.0-30.0 mm) by immunocytochemistry. Com-
pared with the cultured eel, however, PRL cells
were faintly stained, while GH cells were similarly
stained. PRL cells occurred in the rostral pars
distalis in the pituitary (Fig. 1A), separated from
the location of GH cells in the proximal pars
distalis (Fig. 1B). The mid-sagittal section con-
tained less than 5 PRL cells and 10-20 GH cells.
The PRL cells did not form follicular structures, as
typically seen in the adult form of the eel pituitary.
The mean value of the % PRL was 6.5+1.2%,
while the % GH was 15.5+2.0%, 2.4 times higher
than the % PRL (Table 1). Both % PRL and %
GH exhibited a tendency to decrease as lep-
tocephali grew larger (Fig. 2).
The pituitary of the glass eel was morphological-
ly more comparable to that of the cultured eel than
that of the leptocephalus (Fig. 1C, D). The pituit-
Development of PRL and GH Cells in Eel
1063
Fic. 1.
Mid-sagittal sections of the pituitaries in leptocephalus (A, B; 19.7 mm in total length), glass eel (C, D) and
cultured eel (E, F) of the Japanese eel, stained with anti-eel PRL (A, C, E) and anti-eel GH (B, D, F). HT,
hypothalamus; III, third ventricle. Anterior to the left. A and B, X347; C and D, x 126; E and F, x53.
ary in this stage appeared pendent from the
hypothalamus, whereas that of the leptocephalus
was embedded partly in the hypothalamus. The
midsagittal section contained about 30 PRL cells,
gathering at the ventral edge of the rostral pars
distalis, without forming follicular structures (Fig.
1C). The intensity of immunoreaction in PRL cells
were still weaker than that of the cultured eel.
About 40 GH cells cultured in the ventral part of
the proximal part distalis (Fig. 1D). The % PRL
was 12.6+0.5%, significantly higher than that in
the leptocephalus, whereas the % GH (16.7+
1.3%) was similar to that in the leptocephalus
(Table 1).
In cultured eels, PRL cells were located at the
antero-ventral edge of the rostral pars distalis,
mostly forming follicles (Fig. 1E), whereas GH
cells occupied a large part of the proximal pars
distalis (Fig. 1F). The % PRL in cultured eels
(11.8+1.7%) was comparable to that of glass eels.
In contrast, the % GH was significantly increased
tO23:: fae 18 Ze:
1064
TasBL_e 1. Percentages of RPL-cell area (% PRL)
and GH-cell area (% GH) to the whole pituitary
area in different developmental stages of the
Japanese eel
Percentage Developmental stage
of
cell area Leptocephalus
6.5+1.2 (6) 12.6+0.5 (6)**11.8+1.7 (6)*
15.5+2.0 (5) 16.7+1.3 (6) 23.741.8 (6)**
Glass eel Cultured eel
% PRL
% GH
Values represent means+S.E. (n).
* ** and ", significantly different from the values of
leptocephali (*P<0.05, **P<0.001) and glass eels
("P<0.05).
fo)
20
is}
2
w ae, fe)
8
e
32 10 A i
e
e
e
e
(@) —————— ——————
10 20 30
Total length (mm)
Fic. 2. Relationships between percentages of PRL- and
GH-cell areas (% PRL, @; % GH, ©) and body
length in leptocephali of the Japanese eel.
DISCUSSION
In the present study, we examined for the first
time the occurrence and development of PRL and
GH cells in the Japanese eel, using leptocephali as
well as glass eels and sexually immature, cultured
eels. This study was made possible by the success
of collecting a large number of leptocephali and
consequent discovery of the spawaning area of the
Japanese eel by the expedition of the research
vessel Hakuho-Maru [10].
Both PRL and GH cells were detectable in all
the leptocephali examined, even in the smallest
specimen (10.0 mm) which was estimated to be 12
days after hatching. Considering the growth-
promoting action of GH, it is not surprising that
GH cells are present in this early developmental
stage. It is of particular interest, however, that
PRL cells also exist in the stage of the eel living
E. ARAKAWA, T.
KANEKO et al.
offshore, since PRL is supporsed to be the most
important hormone for freshwater adaptation in
euryhaline teleosts [1, 2]. The occurrence of PRL
cells in the early stages of the life has reported in a
marine fish, black sea bream (Acanthopagrus
schlegeli) [14] as well as coho salmon (Oncorhyn-
chus kisutch) [15] and rainbow trout (O. mykiss)
[16]. Our finding and others suggest that PRL is
involved in some other functions than freshwater
adaptation, such as larval growth; in fact it is
generally accepted in amphibian that PRL is in-
volved in larval growth [17].
Both % PRL and % GH similarly decreased as
leptocephali became larger (Fig. 2), implying that
PRL and GH cells develop earlier than other cell
types in the pityuitary. PRL cells were more
faintly stained than those in cultured eels.
Although the intensity of immunocytochemical
reaction does not necessarily reflect the activity of
endocrine cells, PRL cells in leptocephali seem less
active. This is supported by the face that the
number of PRL cells, as well as the % PRL, was
less in the leptocephalus in comparison with GH
cells.
The % PRL increased significantly in the glass
eel compared with that in the leptocephalus,
although PRL cells were still faintly stained when
compared with those in the cultured eel in fresh
water. In cultured eels, the % PRL was similar to
that in glass eels. The increase in the % PRL is
well timed to upstream migration of the glass eel.
Increase in PRL cells prior to migration is also
observed in mullet (Mugil cephalus) {18, 19] and
black sea bream [14]; they migrate from seawater
to brackish water. In black sea bream migrating
from offshore to inshore during the final phase of
postflexion stage, Kimura and Tanaka [14] re-
ported that PRL production was stimulated during
the development from yolk-sac larva to juvenile,
coinciding with their inshore migration. In our
study, leptocephali were collected in the seawater
(34.5%c), while glass eel were collected in the
coastal water with a little lower salinity (30%c); the
difference in environmental salinity is minor.
Thus, the increase of PRL cells in the glass eel
seems to be endogenously prepared in advance for
freshwater adaptation or migration to the river.
The % GH was consistently higher than the %
Development of PRL and GH Cells in Eel
PRL in all the leptocephali examined (Fig. 2), and
the high level was maintained in glass eels. The
abundance of GH cells implies their high cellular
activity, and suggests that the importance of GH
for larval growth and, possibly, osmoregulation in
seawater. The % GH was further increased in the
cultured eel, although they were in fresh water.
The pituitary was distinguishable even in the
smallest leptocephalus, and became morphologi-
cally similar to the adult form of the gland in the
glass eel. In the cultured eel, PRL cells typically
formed follicular structures, the functional signi-
ficance of which is unknown. During early de-
velopmental stages from leptocephalus to glass eel,
however, PRL cells existed only as clusters of cells
without forming follicles, indicating that the
formation of follicular structures occurs in a later
stage, presumably around the period of migration
to fresh water.
In the present study, the activity of PRL cells
seemed to be enhanced in the glass eel, just before
upstream migration. On the other hand, GH cells
remained rather active during all stages of the
leptocephalus and glass eel. These findings are in
consistent with the notion that PRL is responsible
for freshwater adaptation, and GH for seawater
adaptation as well as growth. However, further
studies are required to understand the whole
aspects of osmoregulatory control by PRL and GH
during the early life stages of the eel.
ACKNOWLEDGMENTS
We are grateful to Professor P. P. Hwang, Institute of
Zoology, Academia Sinica, Taiwan, for providing glass
eels. We thank the crew of the Hakuho-Maru and the
scientists aboard. This study was supported in part by
grants-in-aid from the Ministry of Education, Science
and Culture, and also from Fisheries Agency, Japan.
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Hasegawa, S., Hirano, T., Ogasawara, T., Iwata,
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Boeuf, G., Le Bail, R. Y. and Prunet, P. (1989)
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ZOOLOGICAL SCIENCE 9: 1067-1080 (1992)
Innervation of the Caudal-Fin Muscles in the Teleost Fish,
Medaka (Oryzias Latipes)
Yun Isaikawa!
Department of Anatomy, Faculty of Medicine, University of the
Ryukyus, Okinawa 903-01, Japan
ABSTRACT—The peripheral pattern of the spinal nerves in the caudal fin and the spinal motoneurons
that innervate the caudal-fin muscles were described in the adult medaka. The peripheral nerves were
immunochemically stained by using antibodies to neurofilament proteins. To label the spinal
motoneurons retrogradely, horseradish peroxidase was applied to each caudal-fin muscle. The somata
of motoneurons innervating the caudal-fin muscles were distributed over the whole area in the motor
column from spinal segment 25 to 28 of the ipsilateral spinal cord. Most motoneurons were small (6-15
micra in diameter), but larger ones (more than 20 micra in diameter) were sometimes found. The axon
of each motoneuron, rather than entering the nearest ventral root, ran caudad in the anterior funiculus
of the spinal cord for the length of several spinal segments before entering a single ventral root. The
axon that starts from the anterior segment of the spinal cord entered the anterior ventral root. Each
caudal-fin muscle was innervated by several ventral roots of the spinal nerves from 27 to 31B. The
anterior caudal-fin muscles were innervated by the anterior spinal nerves. Thus, motoneurons that
innervate caudal-fin muscles are organized somatotopically along the cranio-caudal axis of the spinal
© 1992 Zoological Society of Japan
cord.
INTRODUCTION
The medaka (Oryzias latipes) is suitable for
observing in detail the nerve pattern in whole
mounts, since the tail regions are thin and almost
transparent in the embryonic stages [1-3].
Moreover, some inbred strains of the medaka have
become available for experimental work [4], and
more than 60 mutant strains including several
morphogenetic ones have been found in the meda-
ka [5, 6]. The first transgenic fish has been
produced successfully in the medaka [7]. Hence,
this teleost fish affords good material for ex-
perimental studies in developmental neurobiology
of vertebrates [see also 8-12].
Anatomical study is an essential step in investi-
gating the development of the nervous system in
the medaka. However, little information on the
neuroanatomy of the medaka is available. The
present study began as an effort to understand how
Accepted June 22, 1992
Received November 6, 1991
' Present address: Division of Biology, National Insti-
tute of Radiological Sciences, Chiba 263, Japan.
the muscle nerves develop in the caudal fin. This
paper describes the anatomy of the nerve-muscle
system in the caudal region of the adult medaka.
Special attention is focused on the innervation of
one of the caudal-fin muscles, the middle interra-
dial muscle (MIR muscle), since its development
will be described in detail in forthcoming papers.
MATERIALS AND METHODS
Materials
The d-rR strain of the medaka (Oryzias latipes)
was given by Dr. H. Tomita (Nagoya University)
in March 1986. The fish of this color mutant strain
shows the wild-type morphology, except for the
mutations in amounts and distribution of several
kinds of pigment cells [5]. The fish have been kept
in our laboratory in plastic aquaria and fed a diet
of Tetra-min (Tetra, West Germany). Adult fish
and young adult fish (15-30 mm in total body
length) of the d-rR strain were used. At least three
fish were used in each labeling experiment and in
each staining (see below). Fry (7-15 mm in total
1068 Y. ISHIKAWA
body length) were also used to show general
structures of the medaka, since the gross patterns
of skeleton, muscles, and peripheral nerves in the
caudal region of the adult fish are almost identical,
except for their sizes, to those of the fry longer
than 7 mm in total body length [2].
Retrograde Labeling with HRP
Adult fish were anesthetized in 0.01-0.03% MS
222 and secured to a plastic plate with a gluey tape.
The unilateral skin above the muscle to be ex-
amined was cut and the underlying muscle tissue
was macerated with a sharpened steel needle.
Small crystals of horseradish peroxidase (HRP,
Toyobo, Osaka, Japan; Lot No. 1417) were put on
the wound. The fish was put in fresh water in such
a way that the head and gill regions were in the
water while the wounded tail region was above the
water surface to avoid dilution or leakage of the
HRP. After 30 min, the fish was washed in fresh
water, released into the balanced salt solution [8],
and allowed to survive for 24—48 hr.
After that, the fish was anesthetized heavily and
killed by decapitation. The caudal part of the body
was put in a solution of 2 or 4% paraformaldehyde
in 0.1 M sodium phosphate buffer (pH 7.4). In the
fixative, the spinal cord (including the proximal
portions of the ventral roots) and the vertebral
column were isolated from the other tissue. The
spinal cord and the vertebral column were fixed in
fresh fixative overnight at 4°C. After washings
over a period of several days with phosphate-
buffered saline (PBS) at 4°C, HRP was histo-
chemically detected in whole mounts by the
methods of Nordlander [13] and Hanker et al. [14].
The spinal cord was cleared with glycerin and
observed in whole mount.
Celloidin Sections of the HRP-labeled Spinal Cord
In order to examine in detail the HRP-labeled
cells in the spinal cord, the celloidin-sectioned
specimens were prepared from the spinal cords
which had been reacted as described above. After
washing in PBS, the reacted spinal cords were
fixed again in 4% paraformaldehyde in 0.1M
sodium phosphate buffer (pH7.4). They were
dehydrated, embedded in celloidin, and sectioned
serially (22 micra in thickness). Nissl staining was
performed in alternate sections by staining with
thionin.
Labeling with Dil
In some caudal-fin muscles, 1,1’-dioctadecyl-
3,3,3’-3’-tetramethylindocarbocyanine perchlorate
(Dil, Molecular Probes, Junction City, Oregon)
was used to label retrogradely the motoneurons
[15]. Young adult fish were fixed in 4% para-
formaldehyde in 0.1 M sodium phosphate buffer
(pH 7.4) for 4h. After washing in PBS, a lesion
was made on the muscle to be examined with a
small scalpel or a sharpened steel needle, and a
small particle of Dil was put on the wound. The
fish was placed in a moist chamber and kept moist
for 10-48 h at room temperature (28°C). After the
Dil was rinsed off in PBS, the spinal cord with the
vertebral column was isolated. It was mounted in
PBS and examined under a fluorescent microscope
equipped with a set of filters appropriate for
rhodamine fluorescence microscopy.
Nerve Staining
Nerve fibers were stained in whole mounts using
anti-neurofilament protein monoclonal antibodies
(all neurofilament proteins, 70 K+ 160 K+210 K,
Maruzen Oil Biochemical or Cosmo Bio Co.,
LTD., Tokyo) according to a previously reported
method [1, 16].
Muscle Staining
The muscle fibers and myotubes were immuno-
histochemically stained in whole mounts using a
monoclonal antibody against chicken troponin T
according to the method of Ishikawa [2].
Double Staining of Muscle and Nerve
The nerve staining and the muscle staining were
combined so that nerve and muscle could be
visualized simultaneously in the same specimens.
The muscle fibers and myotubes were first
reacted with the anti-troponin T antibody and the
HRP-labeled secondary antibody in whole mounts
as described above. They were stained with peroxi-
dase reaction using 3,3’-diaminobenzidine (DAB)
as a substrate. The stained specimens were washed
in 0.1 M glycine-HCl buffer (pH 2.4) overnight at
room temperature (28°C) to remove the anti-
Innervation of Caudal Muscles in Medaka 1069
troponin T antibody and the HRP-labeled anti-
body. The DAB reaction product (brown in color)
remained on the muscle tissue. After washing in
PBS, the nerve fibers of the specimens were then
reacted with anti-neurofilament protein antibody
and the HRP-labeled secondary antibody. They
were stained with peroxidase reaction, this time,
using 4-Cl-1-naphthol as a substrate to obtain a
blue reaction product. The specimens were
cleared with glycerin and immediately observed.
Staining of Acetylcholine Receptors
For observation of neuromuscular junctions,
acetylcholine receptors (AChR) were stained in
whole mounts by the indirect HRP-labeled anti-
body method, using erabutoxin b [17], one of the
curaremimetic toxins from snake venom, and an
anti-erabutoxin b polyclonal antibody [cf. 18].
Nomenclature
We followed Ishikawa [2] in the nomenclature of
B
Fic. 1.
immunochemically using anti-neurofilament protein antibodies.
bones and muscles in the caudal region of the
medaka.
There was a problem in numbering the spinal
nerves since the two cranialmost spinal nerves did
not emerge from the vertebral column but instead,
emerged from the skull in the medaka (see re-
sults). In the present paper these two spinal nerves
were referred to as occipito-spinal nerves accord-
ing to Ray’s description [19] of the cranialmost
spinal nerves of a teleost fish (see refs. [20], [21]
for detailed discussion on the nomenclature of the
rostral spinal nerves in fish; see ref. [22] for an
alternative numbering of the spinal nerves of the
medaka).
RESULTS
Spinal Nerves and Caudal-Fin Muscles
Prior to describing the innervation of caudal-fin
muscles, we provide brief accounts of the ana-
15
Occipito-spinal nerves (OS) and spinal nerves (numbered) of a fry (A and B). The nerves were stained
The left lateral view of the whole-mount
specimen is shown. The head is tilted dorsally. PF, pectoral fin. Scale bar=0.1 mm.
1070
tomical features of the spinal nerves and caudal-fin
muscles.
Two pairs of the occipito-spinal nerves and 32
pairs of the spinal nerves were usually observed in
the medaka (Fig. 1). The occipito-spinal nerve 1,
which is the first postvagal nerve, emerged through
a foramen in the skull. The occipito-spinal nerve
2, the second postvagal nerve, emerged im-
mediately caudal to the skull. Spinal nerve 1, the
third postvagal nerve, passed through a foramen in
the first vertebra. Four cranialmost nerves, name-
ly, the occipito-spinal nerve 1, the occipito-spinal
nerve 2, the spinal nerve 1, and the spinal nerve 2
formed the cervico-brachial plexus at the base of
the pectoral fin. In the caudalmost region, two
pairs of spinal nerves (31A and 31B) emerged
through the foramina in the same caudalmost
vertebra, namely the 31st vertebra.
—
V
Fic. 2.
Y. ISHIKAWA
Figure 2 shows the main branches of a typical
spinal nerve in the tail region. The ventral root
(VR) was larger than the dorsal root (DR), and
extended through its own formen in the vertebra.
Outside the vertebral column the lower part of the
ventral root turned downward to form the ramus
ventralis, while the upper part turned dorsad to
join the spinal ganglion which formed on the
dorsal root. The ramus dorsalis originated near
this ganglion.
The ramus ventralis coursed ventro-caudally in
the ventral region of its own segment and pro-
jected many side branches. The largest side
branch projected laterally from the ramus ventralis
to form a lateral branch at the level of the horizon-
tal septum. The lateral branch ran toward the
lateral surface of the segmental muscle and bifur-
cated dorsally and ventrally near the posterior
RD
SEGMENTAL
MS <—— MUSCLE —-
; {SPINAL
CORD
Drawing of the peripheral pattern of a typical spinal nerve (spinal nerve 25) in the tail region of a fry. The
nerve of the fry were stained immunochemically using anti-neurofilament protein antibodies, and the main nerve
branches were traced. The left lateral view is shown.
A, nerve bundles in the anterior funiculus; DL, nerve
bundles in the dorsal portion of the lateral funiculus; DR, dorsal root; HS, horizontal septum; LB, lateral branch;
MS, myoseptum; RC, ramus communicans; RD, ramus dorsalis; RV, ramus ventralis; SG, spinal ganglion; V,
developing vertebral column, VR, ventral root.
Scale bar=0.1 mm.
Innervation of Caudal Muscles in Medaka
lateral line nerve in the horizontal septum. The
bifurcated branches ran near the anterior myosep-
tum. The ramus dorsalis coursed dorso-caudally in
the dorsal region of its own segment. It received a
small branch (ramus communicans) from the
ramus dorsalis of the next anterior spinal nerve.
The supporting skeleton of the caudal fin was
overlaid with two groups of caudal-fin muscles,
namely, the anterior caudal-fin muscles and the
posterior caudal-fin muscles (Fig. 3).
In the anterior caudal-fin muscles, a pair of deep
dorsal flexors (DDF) and a pair of deep ventral
flexors (DVF) were present in the dorsal and
ventral regions, respectively. There might be
superficial dorsal flexors and superficial ventral
flexors on the surfaces of these two muscles.
However, it was difficult to discriminate between
the caudal superficial muscles and the caudal deep
is
Fic. 3.
1071
muscles. Hence, in the present study, DDF and
DVF are defined as containing in entirety both the
deep and superficial muscle layers. A pair of the
hypochordal longitudinal muscles (HLM) was situ-
ated between the two hypural plates.
In the posterior caudal-fin muscles, 13 to 15
pairs (adult fish) or 8 pairs (young adult fish) of the
interradial muscles were observed beteen the fin
rays. The centrally positioned muscle is called the
middle interradial muscle (MIR muscle). The
MIR muscle was located between the HLM and
the caudalmost pit organ.
Labeling with HRP
In order to estimate the diffusion of HRP from
the wound, we performed a series of experiments
in which HRP activities were reacted in the whole
specimens prior to the isolation of spinal cords.
St passes Ba
Anterior (ANT) and posterior (POST) caudal-fin muscles in a young adult fish. The muscles were stained
immunochemically using anti-troponin T antibody. The left lateral view of the whole-mount specimen is shown.
The vertebrae (V30 and V31) and hypural plates (H1 and H2) are also shown. DDF, deep dorsal flexor; DVF,
deep ventral flexor; HLM, hypochordal longitudinal muscle; LIR, lower interradial muscle; MIR, middle
interradial muscle; UIR, upper interradial muscle; P, caudalmost pit organ. Scale bar=0.1 mm.
1072
From the results, it appeared that HRP did not
diffuse from the site of the application (Fig. 4).
However, in the case of the interradial muscles, we
could not rule out a diffusion of HRP to the same
muscle in the opposite side: In some cases, the
interradial muscles in the opposite side turned
brown after the reaction. In such cases, there were
several lightly stained neurons in the contralateral
sides of the spinal cords that may have been
labeled by a small amount of diffused HRP. In the
present study, therefore, only darkly stained Golgi
like cells were considered to be labeled
motoneurons innervating the HRP-applied
muscle.
Fic. 4.
HRP-positive area in the caudal fin of an adult
fish after applying HRP to the MIR muscle (arrow).
Note that the HRP activity is restricted in the
MIR-muscle region. Left lateral view of the whole-
mount specimen is shown. For abbreviations, see
Figure 3. Scale bar=1 mm.
At least three animals were used for the labeling
experiments of each muscle. The results in each
muscle were generally consistent in all experi-
ments.
Labeled Motoneurons
The somata of the labeled motoneurons were
either spherical or ellipsoidal and found in the
Y. ISHIKAWA
ipsilateral side of the spinal cord (Fig. 5). There
appeared to be two populations which were differ-
ent in the soma size of the labeled motoneurons
(Figs. 6B, 6C and 7), The smaller ones (small
motoneurons, Fig. 6B), to which most of the
labeled motoneurons belonged, were 6-15 micra
in diameter, while the larger ones (large
motoneurons, Fig. 6C) were few in number and
were more than 20 micra in diameter. The anteri-
or caudal-fin muscles (DDF, HLM, and DVF)
were innervated by both the small and large
motoneurons, whereas the posterior caudal-fin
muscles (interradial muscles) were innervated only
by the small motoneurons (Fig. 7).
The labeled small and large motoneurons usual-
ly had two primary dendrites, one running dorso-
rostrally and the other dorso-caudally; both den-
drites ran in the lateral funiculus of the spinal cord,
and branched along their entire courses (Figs. 5B
and 6C). The cranio-caudal extension of the
dendrites was large in the motoneurons filled from
the anterior caudal-fin muscles and small in those
filled from the posterior caudal-fin muscles.
An axon emerged from the ventral portion of
the soma as a single tapering process that extended
ventro-caudally and ipsilaterally in all motoneu-
rons (Figs. 5A and 6A). Axon collaterals were
sometimes observed in the initial part of the axon
(Fig. 6B). The axon did not emerge immediately
via the nearest ventral root, but ran caudad in the
anterior funiculus of the spinal cord for several (2—
4) segment lengths before entering a single ventral
root (Fig. 6A). The axon of the motoneuron that
lies in the more cranial segment of the spinal cord
ran the more ventral course in the anterior funicu-
lus of the spinal cord and emerged from the more
cranial ventral root.
Location of Labeled Motoneurons in the Spinal
Cord
The spinal motor column in the cross plane was
revealed by plotting the locations of the somata of
the HRP-labeled motoneurons in a schematic cross
plane of the spinal cord (Fig. 8). The HRP-labeled
motoneurons filled from each muscle were found
over the whole area of the motor column: No
restricted localization of any specific groups of the
motoneurons was found in the motor column in
Innervation of Caudal Muscles in Medaka
1073
Fic. 5. HRP-labeled motoneurons (arrows) in the adult spinal cords, after applying HRP to the MIR muscle (A) and
the HLM (B and C). The left lateral views of whole mount specimens (A and B), and the cross section (22 micra
in thickness) of the spinal cord embedded celloidin (C) are shown. The cross section is Nissl-stained to show the
gray matter in the spinal cord. The dendrites are indicated by arrowheads. The vertebae are numbered. CC,
central canal; MF, Mauthner fibers. Scale bars in panels A and B=0.1 mm. Scale bar in panel C=20 um.
the cross plane of the spinal cord.
In the sagittal plane of the spinal cord, the
somata of all labeled motoneurons were distrib-
uted from spinal segment 25 to spinal segment 28
of the spinal cord (Fig. 9). Each motor pool of the
anterior caudal-fin muscles stretched over approx-
imately two spinal segments, and each motor pool
of the posterior caudal-fin muscles extended over
about one spinal segment. For example, the motor
pool of the MIR muscle was usually (16/20) lo-
cated in spinal segment 27 and occasionally (4/20)
distributed from spinal segment 27 to the anterior
one-third of spinal segment 28. The motoneurons
were not evenly distributed in a single motor pool
but tended to be clustered (Fig. 5A).
The position of each motor pool was partially
overlapping in the spinal cord (Fig. 9). The motor
pools of the anterior caudal-fin muscles were lo-
cated from spinal segment 25 to spinal segment 27,
while those of the posterior caudal-fin muscles
were distributed from spinal segment 26 to spinal
segment 28; thus, the center of the motor pools of
the anterior caudal-fin muscles was located more
cranially than that of the posterior caudal-fin mus-
1074
Y. ISHIKAWA
B sets
Fic. 6.
C
Drawing of HRP-labeled motoneurons in the adult spinal cords, after applying HRP to the MIR muscle (A
and B) and to the HLM (C). In panel A, the left lateral view of the whole mount specimen is shown. The spinal
nerves (ventral roots) are numbered. In panels B and C, the typical “small” (B) and “large” (C) motoneurons in
cross sections of the spinal cords are shown.
In panels B and C, the axons (large arrows), the dendrites
(arrowheads), the axon collateral (small arrow), the central processes of the spinal ganglion cells (CP), the
anterior funiculus (A), and the lateral funiculus (L) are indicated. CC, central canal; MF, Mauthner fibers. Scale
bar in panel A=0.1 mm. Scale bar in panel B=20 ym, valid for panels B and C.
cles. The number of motoneurons in each motor
pool filled from a single caudal-fin muscle varied
from 1 to 30 (Fig. 9).
Innervation of Caudal-Fin Muscles
By retograde labeling with HRP (Fig. 6A) or
Dil (data not shown), the motor axons that project
to each caudal muscle were revealed. Thus, the
spinal nerves that contribute to the innervation of
each caudal-fin muscle were identified (Fig. 10).
The DDF was innervated by the rami dorsales
from the ventral roots of spinal nerves 28, 29, 30,
and 31 (mainly 30 and only slightly 31). The HLM
was served by the rami ventrales from the ventral
roots of spinal nerves 28, 29, and 30 (mainly 30).
The DVF received the rami ventrales of the ven-
tral roots of spinal nerves 27, 28, and 29 (mainly 28
and 29). All interradial muscles were supplied by
the rami ventrales of the ventral roots of spinal
nerves 30 and 31 (mainly 31).
Muscle Nerve to the MIR Muscle
The peripheral branching pattern of the spinal
nerves was examined in detail in the caudal fin
(Figs. 11A and 11B). A characteristic pattern, the
entire shape of which is like that of a fan-shaped
Fic. 7.
Fic. 8.
Innervation of Caudal Muscles in Medaka
A DDF+HLM+DVF
NUMBER
Oo
15
10
(0) 5 10 15
DIAMETER
Size distribution of labeled somata of the
motoneurons in adult spinal cords, after applying
HRP to the anterior caudal-fin muscles, DDF+
HLM-+DVFE (A) and the posterior caudal-fin mus-
cles, IR (B). All measurements were made on the
drawings of the HRP-labeled motoneurons in cel-
loidin sections and were not corrected for shrinkage.
The average diameter of an individual cell was
calculated as the average of the diameter across the
shortest and longest axis of the soma. The ordinate
indicates the number of motoneurons. The abscissa
indicates the average diameter.
20 pm
MF
Schematic drawing of a cross section of the
caudal spinal cord, showing the motor column
(shaded area) at the right side. The gray matter
(dotted area), central canal (CC) surrounded by
ependymal cells, and Mauthner fibers (MF) are also
shown. A, anterior funiculus; L, lateral funiculus.
NUMBER
Fic. 9.
1075
12-
DDF
BF
4b
8
HLM
AL —_
+—+ a
12 - 7
DVF
8 |
a |e =|
a U6 ]
2
ie + +——_}+_+—_+—_ + = + + |
(5
ZA US :
+——+ +——+—_+ + + + +
A=
4+ U4 :
‘oe eeece Leal
SEGMENT
Histograms showing the distribution pattern of
labeled motoneurons along the cranio-caudal axis of
the caudal spinal cord, after applying HRP to each
caudal-fin muscle of adult fish. A typical result is
shown for each muscle. The ordinate indicates the
number of labeled motoneurons. The abscissa indi-
cates the spinal segments, each of which is divided
into three equal parts; anterior (a), middle (m), and
posterior (p). L1—6, lower interradial muscles; U1-
6, upper interradial muscles. For other abbrevia-
tions, see Figure 3.
1076 Y. ISHIKAWA
Fic. 10. Schematic drawing of muscle nerves to each caudal-fin muscle of the adult fish. The spinal nerves (ventral
roots) are numbered. The main innervating nerves are indicated by thick lines. For abbreviations of muscles, see
Figure 3.
Innervation of Caudal Muscles in Medaka
1077
Fic. 12.
Drawing of the caudal-fin-motor nerve (MN) and the caudal-fin-sensory nerve (SN) in the left lateral view of
the tail region of a young fish. The spinal nerves (rami ventrales) are numbered. The muscle nerve branches in
the MIR muscle are indicated by arrowheads. For abbreviations, see Figure 11. Scale bar=0.1 mm.
tree, was found in this region (Figs. 10 and 12):
The rami ventrales of the ventral roots of several
caudalmost spinal nerves ran ventro-caudally to
form a nerve plaxus at the ventral edge of the
vertebral column. From the nerve plexus, a thick
nerve bundle (caudal-fin-motor nerve) projected
caudad. The caudal-fin-motor nerve ran under-
neath the dorsal part of the HLM toward a space
between two central fin rays.
The caudal-fin-motor nerve bifurcated dorsally
(upper branch of the caudal-fin-motor nerve) and
ventrally (lower branch of the caudal-fin-motor
nerve) at the posterior border of the HLM. The
two nerve branches ran along the posterior ends of
the hypural plates. From these branches, nerve
twigs projected and entered the interradial muscles
to form the neuromuscular junctions (Fig. 11C).
On the other hand, one or two tightly packed
nerve bundles (SN in Figs. 11A and 12) also pro-
jected from the nerve plexus. The nerve bundles
took a course similar to that of the caudal-fin-
motor nerve. However, the distal nerve branches
did not stay in the region of the interradial mus-
cles, but extended further along the fin rays to the
caudal end of the caudal fin. The nerve bundles
were named the caudal-fin-sensory nerve, because
putative sensory axons in the caudal fin should be
contained in the bundles.
Thus, the courses of the muscle nerves to the
interradial muscles were revealed entirely. For
example, the muscle nerve to the MIR muscle
started from the motoneurons in spinal segment 27
of the spinal cord (Fig. 9), extended caudad in the
spinal cord, emerged from the spinal cord via the
rami ventrales of the ventral roots of the spinal
nerves 30, 31A, and 31B, ran further ventro-
Fic. 11.
interradial muscles in an adult fish (C).
Double staining of muscles and nerves in the caudal fin of a young adult fish (A and B) and AChR staining of
The muscles and nerves are immunochemically stained using
anti-troponin T antibody and anti-neurofilament protein antibodies, respectively.
The AChR is stained
immunochemically using erabutoxin b and its antibody. The left lateral views of the whole mount specimens are
shown. In panels A and B, the photographs were taken in slightly different fields at different focus planes. The
neuromuscular junctions in the MIR muscle (MIR) are observed along the courses of the muscle nerve branches
in the MIR muscle (arrowheads). F, central fin rays; LMN, lower branch of the caudal-fin-motor nerve; MN,
caudal-fin-motor nerve; SN, caudal-fin-sensory nerve; UMN, upper branch of the caudal-fin-motor nerve. Scale
bar in panel A=20 ym, valid for panels A and B. Scale bar in panel C=0.1 mm.
1078
caudally for a long distance (about 600 micra), and
finally entered the MIR muscle via nerve branches
of the upper and lower caudal-fin-motor nerves
(Fig. 12).
DISCUSSION
The present study has provided for the first time
an anatomical description of the innervation of the
caudal-finmuscles of the adult medaka. We found
that there was a characteristic pattern of peripheral
nerves in the caudal fin and that the motoneurons
were organized somatotopically along the cranio-
caudal axis of the caudal spinal cord.
The caudalmost musculature in the medaka are
derived ontogenetically from several caudalmost
myomeres [10]. Also, from phylogenetic studies, it
has been pointed out that the caudal-fin muscles of
fish are somitic in origin, being derived from
caudal myomeres [23]. It might be reasonable to
assume, accordingly, that the caudal motoneurons
of the medaka are essentially similar to those
found in the spinal motoneurons innervating the
trunk segmental muscles of the teleost fish.
However, as discussed below, the present results
indicate that not only similarities but also differ-
ences are present between the caudal motoneurons
and the trunk motoneurons.
Innervation and Axonal Pattern
The dorsal caudal-fin muscle, the DDF, is
obviously a caudalmost component of the epaxial
musculature of the trunk muscle system, while the
ventral caudal-fin muscles, the DVF and HLM, are
caudalmost components of the hypaxial muscula-
ture (Fig. 3). The interradial muscles are the
hypaxial muscles as well, because they differenti-
ate in the hypochordal region of the developing
caudal fin [2]. Only DDF is innervated by the rami
dorsales, while the other muscles are served by the
rami ventrales of the ventral roots (Fig. 10). These
observations are consistent with studies of trunk
segmental muscles in the teleost fish; the studies
indicate that the ramus dorsalis and ramus ventral-
is innervate the epaxial musculature and hypaxial
musculature, respectively (see ref. [24] for a re-
view; see also Fig. 2).
In the present study, a motor axon ran longitudi-
Y. ISHIKAWA
nally and caudally for a long distance in the caudal
spinal cord, bypassing several ventral roots before
entering a single ventral root (Fig.6A). This
axonal pattern in the caudal spinal cord differs
from those reported in the trunk spinal cords of the
teleost fish. In the trunk spinal cord of the adult
goldfish, Fetcho [25] showed that most motor
axons ran caudally in the spinal cord and entered
the nearest single ventral root, although some
motor axons bypassed one root before exiting from
the cord. In the rostral trunk region of a larval
teleost fish (zebrafish), Myers [26] reported that a
single axon of each motoneuron entered the
nearest single ventral root, and the motor axons
within the given ventral root were all derived from
motoneuron somata within a single spinal seg-
ment.
Motoneuron Types
The morphology of the trunk motoneurons has
been studied in detail in the adult teleost fish using
modern techniques ({25], [27], [28]; see ref. [24]
for a review). All authors agree that there are two
groups of motoneurons; small motoneurons that
locate in the ventro-lateral portion of the motor
column and larger motoneurons that occupy the
dorso-medial part of the motor column.
Raamsdonk et al. [27] and Fetcho [25] showed
that the differences in the location and morpholo-
gy of the two types of motoneurons were corre-
lated with differences in the functionally different
muscle fiber types they innervate. These investiga-
tors reported that “red motoneurons” which inner-
vate the superficial slow (red) muscles are small in
their soma size and located in the ventro-lateral
part of the motor column; at least some of the
“white motoneurons” which innervate the deep
fast (white) muscle, were larger and lay near the
central canal.
Several authors proposed that the two classes of
motoneurons also had different developmental his-
tories [25, 28]. According to these authors, the
small motoneurons differentiate later than the
larger motoneurons; the small motoneurons and
the larger ones were referred to as “secondary
motoneurons” and “primary motoneurons”, re-
spectively.
In accordance with the above results reported by
Innervation of Caudal Muscles in Medaka
other investigators, two classes of soma size were
found in the spinal motoneurons innervating the
trunk segmental muscles of adult medaka
(Ishikawa, unpublished observations): The small
motoneurons were about 8-16 micra, while the
larger ones were about 20-30 micra in diameter.
In the present study too, we observed that there
were two populations of soma size in the HRP-
labeled motoneurons (the “small motoneurons”
and the “large motoneurons”), although there was
not a very distinct dichotomy of motoneuronal
types (Figs. 6B, 6C and 7).
However, no “large motoneurons” were found
in the motor pools of the posterior caudal-fin
muscles; only the “small motoneurons” were pre-
sent. The “small motoneurons” were located over
all areas of the motor column including the medial
area near the central canal of the spinal cord.
Moreover, the “large motoneurons” in the present
study were distributed in the lateral area of the
cross plane of the motor column (Fig. 6C). Hence,
in the motoneurons of the caudal spinal cord of the
medaka, it seems inappropriate to apply simply the
designation of “white and red” types or “primary
and secondary” types of motoneurons which have
been reported for the trunk motoneurons.
The “large and small” motoneurons in the pre-
sent study may reflect the time sequence of de-
velopment of motoneurons. It is generally
accepted that large neurons are produced before
small ones in any part of the nervous system [29,
30]. The caudal-fin muscles differentiates later
than the trunk segmental muscles [2]. In particu-
lar, the posterior caudal-fin muscles develop much
later than the anterior caudal-fin muscles. If the
motor pools develop in the same sequence as the
groups of muscles, the posterior caudal-fin muscles
would be innervated by the “small motoneurons”.
This hypothesis can explain the fact that only the
“small motoneurons” are present in the motor
pools of the posterior caudal-fin muscles.
ACKNOWLEDGMENTS
This paper is dedicated to my late father, Kazuo
Ishikawa, who had warmly supported my interest in
science. I am grateful to Dr. Hideo Tomita for supplying
the medaka. I also thank Mr. Yoshinari Fusa and Miss
Katsumi Chinen, for their technical assistance. This
1079
study was supported by a Grant-in-Aid for Scientific
Research on Priority Area (molecular basis of neural
connection), Ministry of Education, Science and Cul-
ture, Japan.
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Anat. Embryol., 167: 125-139.
Westerfield, M., McMurray, J. V. and Eisen J. S.
(1986) Identified motoneurons and their innervation
of axial muscles in the zebrafish. J. Neurosci., 6:
2267-2277.
Kanemitsu, A. (1971) Relation entre la taille des
neurones et leur €poque d’apparation dans la moelle
épiniére chez le poulet; Etude autoradiographique
et caryométrique. Proc. Japan Acad., 47: 432-437.
Jacobson, M. (1978) Developmental Neurobiology.
Plenum Press, New York and London, 2nd ed.
ZOOLOGICAL SCIENCE 9: 1081-1085 (1992)
[COMMUNICATION]
© 1992 Zoological Society of Japan
Sexual Development of Immunocompetence
in the Toad, Bufo regularis
ABDEL HAKIM SAAD
Zoology Department, Faculty of Science,
Cairo University, Cairo 12613, Egypt
ABSTRACT—The kinetics of primary anti-rat erythro-
cytes (RRBC) antibdody were compared in juvenile and
adult toads Bufo regularis. The antibody titers were
measured by haemagglutination assay, and antigen spe-
cific rosette-forming cells were enumerated in the spleen
using immunocyto- adherence assay. Both adults and
juveniles responded to RRBC, with adult being always
the highest responder. Among the adult toads, slight
differences between male and female could be observed.
However, juvenile toads did not demonstrate sex-
associated immune differences.
INTRODUCTION
Immunocompetence is a complex physiological
process that develops during ontogeny and is
maintained throughout animals’s life cycle. Anu-
ran amphibians are excellent models for immuno-
logic studies from the perspectives of both evolu-
tionary and developmental biology [1, 2]. In
comparison with amniotes, there is an obvious
advantage in using the amphibian models for
studying the ontogeny of immune responses, since
amphibians develop as free-living embryos and
larvae thus allowing experimental manipulation
from the earliest stages of immune system develop-
ment [3, 4]. However, little attention was paid to
the immune response of anuran amphibians during
sexual maturity and ageing [5, 6]. Here, we
examine the importance of major biological vari-
ables such as sex and age on the humoral immune
response of the toad, Bufo regularis. In addition,
Accepted July 4, 1992
Received January 7, 1992
the role of sex hormones played in the immune
response of amphibians is discussed.
MATERIALS AND METHODS
Animals and housing Male and female toads,
Bufo regularis (Anura, Bufonidae), were obtained
from Abu Rawash area in the vicinity of Cairo
(Egypt). Toads were kept in glass aquaria with tap
water in a sunny animal room at ambient tempera-
ture of 20-27°C during spring and provided with
granulated trout feed ad libitum.
Cell preparation Individual spleens were ex-
cised carefully and gently teased apart with fine
forceps over stainless-steel meshes in ice-cold,
serum-free Leibovitz (L-15) medium (CIBCO,
Grand Island, N.Y., USA). Tissue debris were
decanted, and cells were washed twice with ice-
cold medium by centrifugation (400 xg, 5 min). In
the last wash, pellets were counted and their
viability was assessed by the trypan blue exclusion
test.
Histological procedure After dissection, the
spleen was fixed in 10% buffered formalin, dehy-
drated in alcohol and embedded in paraffin. Eight
yem-thick sections were stained with haematoxylin
and eosin.
Antigen and immunization Rat erythrocytes
(RRBC) were obtained from at least three ani-
mals. Pooled heparinized blood was centrifuged
1082
(400g, 5 min) and the cells were washed thrice
with phosphate-buffered saline (PBS), pH7.2
before use. Preliminary experiments disclosed that
0.4 ml of 10% RRBC is an optimum dosage which
induced maximum antibody (Ab) response when
given intraperitoneally (i.p.). Control unimmu-
nized toads were injected i.p. with 0.4 ml PBS, pH
7.2. Immunized and control blood samples were
allowed to clot for 2 hr at room temperature and
then overnight at 4°C. After centrifugation (650 x
g, 15 min), individual sera were heat-inactivated at
56°C for 30 min and stored at —20°C until use.
Assay of humoral immune response Groups of
animals were decapitated at specific intervals after
immunization and a single spleen cell suspension
was prepared as described previously. Antigen
specific rosette-forming cells (RFC) were enumer-
ated in the spleen using immunocyto-adherence
assay as described by Saad and Ali [7]. On the
basis of actual number of cells for each sample, the
RFC per 10° spleen cells was computed. Serum Ab
titers were determined in standard haemagglutina-
tion assay as described previously [7]. Ab titers
were expresed as log reciprocal of the highest
dilution showing microscopic agglutination.
Statistical analysis Student t-test was used to
determine levels of significance between control
and experimental groups. Diferences were consi-
dered to be significant at P values <0.05.
RESULTS AND DISCUSSION
Field studies have shown that the growth rate of
anuran amphibians, as determined from snout-
vent (SV) length, can be used as an indicator of
growth and sexual maturity [8, 9]. As depicted in
Figure 1, the total number of splenic lymphocytes
recovered from males and females appear to
increase with growth. At SV-length greater than
95mm, the lymphocyte population starts to de-
cline, possibly reflecting an increase in animal age
and approaching senescence. However, no sex-
related differences were observed between male
and female toads.
comparatively the variations in the kinetics of
immune response between two different SV-
For simplicity, we analyzed
A. H. Saap
(Xx 10°)
- Yj, %2
= 1
e mM ¢¢
)
[e)
Ios
jae
=
>
—_
6
Ww
(e)
Ow 4
Lu
oo
=
= 2
(30 -55) (60-75) (80-95) (> 95)
SNOUT- VENT LENGTH (mm)
Fic. 1. Total number of viable splenic lymphocytes
obtained from female and male toads, B. regularis of
varying snout-vent length. Each column represents
the mean number of 10-15 separate animals. Ver-
tical bars indicate standards error of the mean.
length: 30-55 mm (i.e. juvenile toads) and 80-95
mm (i.e. adult toads). The main issue raised in our
study is that the response to RRBC in B. regularis
appeared to be highly age-dependent but not
sex-dependent. Juvenile animals showed a lower
RFC and Ab titers. A considerable number of
RFC were detected in male and female toads on
the first four days after RRBC injection. Thereaf-
ter, a sharp decline occurred in the number of RFC
during the subsequent periods. The shape of the
curves was otherwise quite similar (Fig. 2A).
However, despite the fact that the peak of RFC
response to RRBC was somewhat delayed and
diminished in adult males in comparison with adult
females, yet sex-associated differences at some
time point were not significant (Fig. 2B). The
kinetics of the primary anti-RRBC Ab response
are depicted in Figure 2. It can be seen that, in
adult toads, primary response appeared to be more
vigorous, and total Ab titers were significantly
higher (P<0.001) than those of juvenile toads
(Fig. 3A). Also, with adult female toads the peak
of primary Ab response to RRBC is somewhat
delayed in comparison with adult male toads (Fig.
3B).
It is concluded that juvenile toads have the
competence to respond to RRBC but their humor-
Sex, Age and Immunity of Toads 1083
(x104)
NUMBR OF RFC
Oo 4 8 12 16
DAYS POST IMMUNIZATION
Fic. 2. Kinetics of rosette-forming cells (RFC) response in the spleen of female (@—@) and male (O—©) toads, B.
regularis. Juvenile (A) and adult (B) toads were immunized on day O with 0.4 ml 10% RRBC suspension. Each
point represents the mean response of 3-5 separate animals and the vertical bars indicate standard error of the
mean. =0.05<P<0.01 and NS=not significant.
RECIPROCAL 10g, TITERS
oa Ts) 12 BS C20 Oars ia iG) 20
DAYS POST IMMUNIZATION
Fic. 3. Mean serum haemagglutinin titers in female (@—@) and male (O—©) toads, B. regularis: Juvenile (A) and
adult (B) toads were immunized on day O with 0.4 ml 10% RRBC suspension. Each point represents the mean
response of 3-5 separate animals and the vertical bars indicate standard error of the mean. *=0.05< P<0.01
and NS=not significant.
al immune system is still not full grown. Taken RRBC depends on the maturational status of the
together, these observations present the possibility spleen, which in juvenile toads has a poorly
that the maturation of the immune response to defined histology, relatively low number of lym-
1084 A. H. Saab
a A te
os athe , —
Fic. 4. The spleen of B. regularis sacrificed during mid-spring. (A) Spleen of adult toads with the thickened wall of
the central arteriole which is densely populated with lymphoid cells.
(B) Spleen of juvenile toads with a
considerable number of lymphoid cells which are present in the red and white pulp. Scale bar: 100 um.
phoid cells, and small mass. Histologically, the
spleens were large in adult toads, and their white
pulp was filled with numerous lymphocytes aggre-
gated around the blood vessels (Fig.4A). In
contrast, the spleens were smaller and significantly
lighter in juvenile toads and the lymphocyte
aggregations were diminished (Fig. 4B). Immuno-
logical maturity, therefore, attained only when the
pool of immunocompetent cells in the spleen
becomes sufficient in size and diversity.
The data reported here, which suggest the
assumption of sexual maturity may relate to
greater reactivity to RRBC, are also of interest
when considering hormonal systemic controls on
immune regulation. The shift in immune reactivity
with sexual maturity in B. regularis should be
Sex, Age and Immunity of Toads
viewed in the context of the data which suggest
constancy at a lower reactivity level in sexually
immature juveniles [5, 6]. In marine teleosts, the
humoral activity of mature females to sheep
erythrocytes (SRBC) was lower than males or
immature females in the spawning season [10].
Response to SPRC in partridges was found to be
sex-independent, but declined markedly in par-
tridges older than l-years [11]. Other studies on
pigeons have demonstrated that maturation of the
immune functions in terms of anti-SRBC rosette
formation and migration inhibition kinetics occurs
at 3-4 months of age, which coincides with the
onset of sexual maturation [12]. Therefore,
although the evidence is circumstantial, the
changes found in the activity of B. regularis to
RRBC could be imputed to the influence of sex
hormones. It is clear from our study that during
spring, i.e. the time of sexual maturation in
amphibians of temperate regions [13, 14], the
immunologic responses investigated are different
between juvenile and adult animals. Indeed,
before sexual maturation, the humoral responses
are similar between the sexes. It seems that the
onset of the reproductive maturity triggers some
differential capacity in the immune reactivity
between juvenile and adult toads. This observa-
tion alone unequivocally supports the role of sex
hormones in the immune expression. However,
the mechanisms by which sex hormones control
the immune response are not known. It is also not
known whether testosterone and estradiol have
opposing effects on the same cells or whether each
hormone acts on different arms of the regulatory
1085
circuits of the immunologic network [15, 16].
REFERENCES
1 Du Pasquier, L. (1976) In “Comparative Immuno-
logy”, Ed. by J. J. Marchalonis, Blackwell Scientific
Publ., Oxford, pp. 390-419.
2 Katagiri, C. and Tochinai, S.
Growth and Differ., 29: 297-305.
3 Flajnik, M. F., Hsu, E., Kaufman, J. F. and Du
Pasquier, L. (1987) Immunology Today, 8: 58-64.
4 Ruben, L. N., Clotheir, R. H., Horton, J. D. and
Balls, M. (1989) Bio Assays, 10: 7-12.
5 Wright, R. K. and Cooper, E. L. (1980) In
“Development and Differentiation of Vertebrate
Lymphocytes” Ed. by J. D. Horton, Elseriver,
North Holland, pp. 141-152.
6 Ruben, L. N., Clotheir, R. H., James, H. S. and
Balls, M. (1984) Cell Differentiation, 14: 1-5.
7 Saad, A.-H. and Ali, W. (1992) Zool. Sci., 9: 349-
356.
8 Ryan, R. A. (1953) Copeia, 1953: 73-80.
9 Hagstorm, T. (1977) Zool. Scripta, 6: 61-68.
10 Nakanishi, T. (1986) Vet.
pathol., 12: 336-342.
11 Randi, E., Chiricolo, M., Spagesi, M., Ghendini, I.,
Savigni, G., Giovannini, A. and Franchesi, C.
(1985) Dev. Comp. Immunol., 9: 679-690.
12 Selvaraj, P. and Pitchappan, R. M. (1988) Dev.
Comp. Immunol., 12: 379-384.
13 Lofts, B. (1984) In “Marchall’s Physiology of
Reproduction”. Ed. by G. E. Lamming, Churchill
Livingstone, Edinburgh, Vol. I, pp. 127-305.
14 Moore, E. L. and Zoeller, R. T (1990) Horm.
Behav., 13: 207.
15 Forsberg, J. G. (1984) Arch. Toxicol., 55: 79-87.
16 Grossman, C. J. (1984) Endocrin. Rev., 5: 435-455.
(1987) Develop.
Immunol. Immuno-
Co
ZOOLOGICAL SCIENCE 9: 1087-1092 (1992)
[COMMUNICATION]
© 1992 Zoological Society of Japan
Electron-Microscopic Observation of an Ectopic PGC-like
Cell in the Teleost Oryzias latipes
HIROKUNI KOBAYASHI and Tomi-o HISHIDA
Department of Biology, School of Liberal Arts, Asahi University,
1851-1 Hozumi-cho, Motosu-gun, Gifu 501-02, Japan
ABSTRACT— In one specimen of newly hatched fry of
the medaka, Oryzias latipes, one primordial germ cell
(PGC)-like cell was found by light microscopy in the
subepidermal layer of the body wall lateral to the body
cavity; an ectopic site far from the gonad where the
normal primordial germ cells (PGCs) were actually
located. The ultrastructures of the ectopic PGC-like cell
was examined by electron microscopy, and compared
with those of the normal PGCs in the gonad in the same
specimen.
It was demonstrated that the above stated ectopic
PGC-like cell, though spheroidal in shape, had striking
morphological similarities without any degenerative
aspect to the normal PGCs in the gonad at the
ultrastructural level, especially due to the presence of
two peculiar organella, i.e., the germinal dense bodies
(GDB) and various lengths and forms of the sheets of
agranular reticulum (AR).
The present evidence suggests that the ectopic PGC-
like cell in the body wall might be one of the PGCs which
had settled down in the aberrant site during migration
into the gonadal region in the early stages of
embryogenesis, and that the PGCs or their presumed
forerunners, even in the ectopic site, could differentiate
themselves at least to the same developmental stage as
that of the normal PGCs actually located in the gonad at
the time of hatching.
INTRODUCTION
In various animals including vertebrates and
invertebrates, it has been fully confirmed that
primordial germ cells (PGCs) segregate from the
somatic cells during the early stages of develop-
ment extra-gonadally or extra-embryonally, and
Accepted June 23, 1992
Received March 22, 1992
then migrate through various tissues towards their
final location as the ultimate gamates in the
gonadal anlagen [1]. The occurrence of the PGCs
in various ectopic sites after the completion of
their migratory journey into the gonadal anlagen
has been reported in embryos of several species of
vertebrates [2]. On the other hand, persistence
and differentiation of the ectopic PGCs past the
embryonic period have been reported only spor-
adically by Stolk [3], Hardistry [4], and Zamboni
and Upadhyay [5]. Recently in the field of the
study of germ cell sex differentiation in the
medaka (Oryzias latipes) using light microscopy,
we encountered with a fortunate oppotunity to
note the presence of one ectopic PGC-like cell
within the cell layer constituting the lateral body
wall, together with the normal PGCs in the gonad
in the same plane of an epon-embedded section of
One specimen of newly hatched fry. Thus far,
ectopic PGCs or PGC-like cells in the medaka,
those during migration before hatching have been
reported by Gamo [6], while never reported those
after hatching when the normal PGCs are located
already in the gonad. In the present work,
therefore, a preliminary attempt was made to
elucidate whether or not the ectopic PGC-like cell
in the body wall, though only one cell, has the
same ultrastructural features as those of the
normal PGCs in the gonad in the same specimen of
fry by using electron microscopy.
MATERIALS AND METHODS
The orange-red strain (d-rR) of the Japanese
1088
medaka (Oryzias latipes) was used. Under anes-
thesia in 0.015% phenylurethane aqueous solu-
tion, newly hatched fry within a day after hatching
were dissected to remove the head and tail. The
remaining trunk regions including the gonad were
prefixed in a modified Karnovsky’s fixative con-
taining 0.1% picric acid [7] for 4-5 hr at room
temperature. After rinsing in 0.1M cacodylate
buffer (pH 7.3) at 4°C, they were postfixed in 1%
OsO, in the same buffer for 1-2 hr at 0°C. The
fixed materials were dehydrated in a series of
graded ethanol and followed by embedding in a
Spurr’s resin (Taab). Both thick and thin sections
were cut transversely against the body axis of the
fry with a diamond knife (Diatome) on a Porter-
Blum MT-2B ultratome. The former were stained
with toludine blue for light microscopic observa-
tion. The latter were double-stained in uranylace-
tate and lead nitrate, and examined by a JEM-100
U electron microscopy at 80 kv.
H. KOBAYASHI AND T. HISHIDA
RESULTS AND DISCUSSION
At the time of hatching, most fry had rather
small gonads shortly after formed. These gonads
were still sexually indifferent and contained a few
developmentally undifferentiated large PGCs,
each enveloped by thin strands of the somatic cells.
In one such specimen of the fry, an ectopic
PGC-like cell was seen embedded compactly
within a relatively thick subepidermal cell layer
just beneath the outermost thin epithelium of the
lateral body wall, together with the normal PGCs
in the gonad (Figs. 1 and 2A, B). Similar ectopic
PGC-like cells could not be detected in another
following sections of the remaining parts of the
present specimen as far as examined. A basement
membrane was evident below the subepidermal
layer inlcuding the PGC-like cell (Fig. 1, arrow-
heads and Figs. 2A, B). However, the muscular
layer did not developed yet between the parietal
and the body wall layers. The PGC-like cell
Fic. 1.
Low-magnified electron micrograph of a cross section of the posterior trunk region including an ectopic
PGC-like cell a in the body wall and two normal PGCs b, c in the sexualy indifferent gonad of a newly hateched
fry, Oryzias latipes. G, gut; BC, body cavity; BW, body wall; PL, parietal layer; BM, basement membrane
(arrowheads). Bar, 5 ~m.
Fic. 2.
A. Electron micrograph of the ectopic PGC-like cell shown as a in Figure 1. B. Another section of the same
cell, in which a large, voluminous nucleus with a prominent nucleolus can be seen. Each insertion at the bottom
right is a magnification of the encircled area in either A or B, showing the desmosomal connection. Arrowheads
indicate occasional, small cytoplasmic protrusions from the cell surface. AR, sheets of agranular reticulum (short
arrows); GDB, germinal dense bodies (asterisks); E, epidermal layer; SE, sub-epidermal layer; N, nucleus; Nu,
nucleolus; AL, annulate lamella; BM, basement membrane. Long bar, 2 “m. Short bar, 0.2 um.
Fic. 3.
A and B are respectively electron micrographs of two normal PGCs, b and c in the sexually indifferent gonad
in Figure 1. SC, enveloping somatic cells; GDB, geminal dense bodies (asterisks).; AR, sheets of agranular
reticulum (arrows); AL, annulate lamella; BM, basement membrane. Bar, 2 «m.
1089
like Cell in Oryzias
Ectopic PGC
Fic. 2.
H. KoBAYASHI AND T.
HISHIDA
Ectopic PGC-like Cell in Oryzias
appeared somewhat oval in shape, as compared
with the normal PGCs in the gonad (Figs. 3A, B),
and showed no trace of degenerative appearance.
The maximum diameter was about 20 ~m and the
minimum was about 15 um, the averaged value of
which was near to that of the normal PGCs in the
gonad. The nucleus was also large and spherical in
outline with a prominent nucleolus (Fig. 2B) as
usual for that of the normal gonadal PGCs (not
shown). The cytoplasmic membrane of the PGC-
like cell was in extensive close contact with that of
the surrounding subepidermal cells, but no specific
relationship such as desmosomes were observed.
In contrast, desmosomes and sometimes anasto-
mosing structures were detectable between the
membranes of neiboring cells of either the
epidermal or the subepidermal layer, and between
those of both layers (Figs. 2A, B., rectangles). On
the cell surface of the ectopic PGC-like cell, tiny
cytoplasmic processes of various forms were seen
protruding into the surrounding cells (Figs. 2A, B,
arrowheads). It is uncertain if such pseudopod-
like cytoplasmic processes are involved in active
migration of the ectopic PGC-like cell during its
own amoeboid mobility. In general, passive
migration of the PGCs through the morphogenic
movement by the surrounding tissues has been
proposed in the medaka (O. lateipes) by Hogan [8]
and Hamaguchi [9].
Two characteristic structures that had been first
described by Hogan [8] as specific cytological
markers for unequivocal identification of the PGCs
in the medaka (O. latipes) were clearly discernible
in the cytoplasm of the ectopic PGC-like cell. One
of these was the clumps of germinal dense bodies
(GDB) of granular structure sometimes with or
without the association of mitochondria (Figs. 2A,
B., asterisks). Similar, detailed descriptions of the
GDB in the medaka (O. latipes) has been pre-
sented first by Satoh [10] and later by Hamaguchi
[9] and Kanamori ef al. [11]. The other character-
istic structure was the long sheets of agranular
reticulum (AR) which often ran in the cytoplasm
parallel to the curvature of the nucleus (Figs. 2A,
B., short arrows). Occasionally, shorter or loop-
forming segments of the sheets were also seen.
Unlike such a smooth membraneous structure,
poorly stacked, short-linked annulate lamella
1091
(AL) were detectable infrequently. All of these
structures, GDB, AR, and AL, could also be
observed similarly in the gonadal PGCs (Figs. 3A,
B).
As mentioned above, the present observation at
the ultrastructual level revealed that the ectopic
PGC-like cell in the body wall had almost identical
morphological features to those of the normal
PGCs in the gonad, principally the two peculiar
cytological markers noted above. It may be sure
that the present observation is the first demonstra-
tion of the ultrastructure of the ectopic-PGC like
cell in fish, at least in the medaka.
In the present work, the occurrence of the
ectopic PGC-like cell in the mesoderm-derived
subepidermal layer of the body wall probably
indicates the migratory route rather than the origin
of the PGCs. In the medaka, PGCs have been
predominantly referred to as having an endoder-
mal origin since they are first recognizable on the
peripheral endoderm at the neurula stage, accord-
ing to Hogan [8] and Hamaguchi [9]. More
recently, however, Timmermans and Taverne [12]
injected *H-thymidine into embryos at an early
cleavage stage in the teleost, Barbus conchonius,
and demonstrated that the presumptive PGCs
segregate from the somatic cells between mid
epiboly and late epiboly, before the three germ
layers have been formed. This result well coin-
cides with that of Gamo’s light-
microscopical studies in the medaka [12]. There-
fore, it is most likely that the ectopic PGC-like cell
in the present work is one of the PGCs or their
forerunners which has settled down in the extra-
gonadal tissues during migration. Moreover, it
may be suggested that the presumed forerunners
of the PGCs in the medaka, which must have
segregated from the somatic cells during the early
stages of embryogenesis, could differentiate them-
selves without any degeneration, even in the
ectopic site, at least to the stage of becoming the
identifiable PGCs with typical features in the
gonad at the time of hathcing.
Postnatal differentiation of the ectopic PGCs
has been reported in lampreys by Hardisty [3], in
several species of adult fish by Stolk [4], and in
mice by Zamboni and Upadhyay [5]. In all these
works, it is very interesting that the ectopic PGCs
earlier
1092
were demonstrated to survive for a certain pro-
longed period while differentiating as oocytes. It
remains to be elucidated whether or not such
similar phenomenon as above might occurr as for
the ectopic PGCs in the medaka. For further
study, many more examples of the ectopic PGCs in
the medaka during or following embryogenesis will
be needed.
ACKNOWLEDGMENTS
We are grateful to Dr. K. Ikenishi of Osaka City
University for his valuable advice and discussion during
this study, and also to Dr. E. Haig for his reading and
correction of the manuscript.
REFERENCES
1 Niewkoop, P. D. and Sutasurya, L. A. (1979)
Primordial germ cells in the chordates. Cambridge
Univ. Press, Cambridge, pp. 71-127.
2
H. KosayAsHI AND T. HISHIDA
Jirasek, J. E. (1976) Principles of reproductive
embryology. In “Disorders of Sexual Differentia-
tion”. Ed. by J. L. Simpson, Academic Press, New
York, pp. 51-110.
Stolk, A. (1958) Nature, 182: 1241.
Hardisty, M. W. (1965) J. Zool., 146: 305-387.
Zamboni, L. and Upadhyay, S. (1983) J. Exp.
Zool., 228: 173-193.
Gamo, H. (1961) Jpn. J. Zool., 13: 101-115.
Ito, S. and Karnovsky, M. J. (1968) J. Cell Biol., 39:
168.
Hogan, J. C. (1978) J. Ultrastruct. Res., 62: 237-
250.
Hamaguchi, S. (1982) Cell Tissue Res., 227: 139-
151.
Satoh, N. (1974) J. Embryol. Exp. Morphol., 32:
195-215.
Kanamori, A., Nagahama, Y. and Egami, N. (1985)
Zool. Sci., 2: 695-706.
Timmermans, L. P. M. and Taverne, N. (1989) J.
Morphol., 202: 225-237.
ZOOLOGICAL SCIENCE 9: 1093-1096 (1992)
[COMMUNICATION]
© 1992 Zoological Society of Japan
Dependence of Prolactin-Stimulated Tail Fin Growth and Molting
on Water in Male Salamanders (Hynobius nigrescens)
Masato HAsumi and HISAAKI IWASAWA
Biological Institute, Faculty of Science, Niigata University,
Niigata 950-21, Japan
ABSTRACT—Prolactin (PRL)-stimulated tail fin
growth and molting are dependent on water in male
Hynobius nigrescens. Tail length and height increased in
PRL-injected groups regardless of habitat (1.e., terrest-
rial or aquatic), but the aquatic habitat helped the rapid
growth of tail fin. Even under low-temperature condi-
tions (mimic breeding season), molting was speeded up
in all saline-injected animals housed in a fully aquatic
habitat, and further speeded up by PRL regardless of
habitat, suggesting that an aquatic environment induced
the secretion of PRL in this species.
INTRODUCTION
It is known that in Triturus cristatus carnifex [1],
Notophthalmus viridescens [2], and Cynops pyr-
rhogaster [3] prolactin (PRL) increases tail height;
but we do not know whether PRL stimulates an
increase in tail length, perhaps due to difficulty in
comparing tail lengths. We [4] observed that in
Hynobius lichenatus many individuals possessed
variously regenerated tails, suggesting that the tail
breakage occurs in other tail-unautotomizable
species: tail length may be valid when making a
comparison of its change in the same individual.
So we added in this study tail length data to
PRL-stimulated parameters using animals housed
individually. Also, in winter-dormant male H.
nigrescens kept in a terrestrial habitat, tail height
does not show any noticeable increase following
the administration of SIU PRL [5]. This first
Accepted June 18, 1992
Received November 27, 1991
suggests that the absorption of water from an
aquatic environment is necessary for the growth of
tail fin. To examine this possibility, we conducted
an experiment in a fully aquatic or terrestrial
habitat.
Molting in amphibians usually occurs every few
days and is speeded up at higher temperatures [6].
It is known that in urodeles molting is induced by
thyroid hormone and/or PRL [1, 7]. However, a
difference in molting according to habitat (i.e.,
terrestrial or aquatic) is unknown. We also report
here water-dependent (without PRL) or habitat-
independent (with PRL) molting in H. nigrescens
under low-temperature conditions.
MATERIALS AND METHODS
Adult male H. nigrescens were collected from a
pond in Iwamuro-mura, Niigata Prefecture, Japan
(37°44°N, 138°50’E; 180m elevation) during the
breeding season of March 1989. After emergence
from the water they were raised in large, shaded
terraria located outdoors at Niigata University and
provided with terrestrial isopods and earthworms
as food.
On 18 July, the animals maintained were trans-
ferred from the terraria to a room regulated at 8°C,
and here experiments were conducted from 21 July
to 8 August. They were not fed during this period.
A temperature of 8°C was settled as a mimicry of
the breeding season. The males used were sea-
sonally immature [8]. They were divided into four
groups (n=7/group): (1) aquatic-saline (AS), (2)
1094
aquatic-PRL (AP), (3) terrestrial-saline (TS), and
(4) terrestrial-PRL (TP). In groups AS and AP,
the animals were enclosed individually in a plastic
box (21 cm long, 14 cm wide, and 3.5 cm deep with
a lid) nearly filled with aged tap water; and in
groups TS and TP, in a box with a moist paper
towel. The animals were injected intraperitoneally
with a saline solution (0.6% NaCl aq.) in groups
AS and TS, or 5IU ovine PRL dissolved in the
saline solution in groups AP and TP every other
day. The PRL (Sigma Chemical Co., St. Louis)
used had a mean potency of 311U/mg. The
volume of fluid injected was always 0.1 ml. Prior
to the injection, the animals were anesthetized
with 0.1% MS 222 solution, weighed to the nearest
0.1 g with a Roberval’s balance, and measured to
the nearest 0.1mm with slide calipers. The
following data were recorded: snout-vent length,
measured from the tip of the snout to the posterior
angle of the vent; tail length, from the posterior
angle of the vent to the tip of the tail; head width,
at the broadest part; and tail height, at the highest
part. The experimental animals were brought back
to their own box immediately after the injection,
and awoke from the anesthesia in approximately
0.5-2 hr. The occurrence of molting was moni-
tored at the time of injection.
If the data taken in a parameter showed a
meaningful change (i.e., increase or decrease), the
paired f-test was used for a comparison between
initial and final values in that parameter (alpha=
0.05).
TABLE |.
M. Hasumi AND H. Iwasawa
RESULTS AND DISCUSSION
Snout-vent length, body mass, and head width
did not show a meaningful change in any group. A
rapid gain in body mass occurs due to water uptake
and head width increases greatly after entering a
breeding pond in male H. nigrescens [9]. We were
unable to induce these phenomena in seasonally
“immature” males with the administration of PRL.
However, PRL stimulates an increase in head
width in some of seasonally “mature” male H.
nigrescens [5].
In groups AS, AP, and TP, tail length and
height increased significantly, but not in group TS
(Table 1). Their increase in group AS was very
small. In all in group AP and some in group TP,
the tail faintly assumed a fin-like appearance. The
basal and dorsal part of the tail stood out a little in
these animals. In group AP, tail length and height
increased linearly and extremely until the tenth
day after the beginning of injection, and thereafter
plateaued (Fig. 1). In group TP, they increased
slowly from the sixth day until the end of the
experiment. The rate of increase in tail height in
group AP was nearly twice as great as that in group
TP at the end of the experiment. Increased and
plateaued tail height in group AP was not so great
as that seen in typical aquatic phase males during
the breeding season, and the rate of increase in tail
height in group AP was similar to that at the
transition between females before and after enter-
ing the water [9]. Kikuyama ef al. [3] stated
concerning C. pyrrhogaster that the simultaneous
administration of estradiol blocked the PRL-
induced increase in tail height. That is, PRL
Changes in tail length and height (mean+SE) in groups aquatic-saline (AS),
aquatic-prolactin (AP), terrestrial-saline (TS), and terrestrial-prolactin (TP) at 8°C, as
shown by initial and final (in parentheses) values
Group n Tail length (mm) P Tail height (mm) P
AS 7 So muctenlaO) 0.001 7.9+0.2 0.02
(Sak tlt) (8.1+0.2)
AP 7 OA 22 0.001 8.0+0.2 0.001
(60.4 +2.6) (9.0+0.2)
TS il 61.4+2.0 NS 8.1+0.1 NS
(61.5+2.0) (8.0+0.1)
TP 7 51.4+2.8 0.001 8.2+0.2 0.01
(G5:2==331)) (8.7+0.2)
Tail Fin Growth and Molting in Salamanders
is 5
|
\
x:
¥
\
z
Se
*
% CHANGES IN TAIL LENGTH AND HEIGHT
4
vin 1-4 a
: of fouaet
— SA
Co) eaaiek
(0) 2 4 6 8 10 12 14 16 18
DAYS
Fic. 1. Prolactin-stimulated increase in tail length (solid
circle) and height (open circle) in groups AP and TP
(see the caption of Table 1) at 8°C. Vertical bars
denote SE on either side of the mean.
increases tail height in the male, but not in the
female because of the higher concentration of
estradiol. In H. nigrescens, however, plasma
concentrations of estradiol are at the base in
females both before and after entering a breeding
pond (Hasumi, Iwasawa, and Nagahama; unpub-
lished data) when tail height increases only a little
[9]. These lowest concentrations of estradiol
therefore suggest that androgen is involved in the
greater growth of male tail fin (sexual dimorphism)
of this hynobiid species, although PRL alone
increases tail height in several salamandrids [1-3].
An extremely high concentration of testosterone is
determined in male H. nigrescens in the process of
tail fin growth (Hasumi, Iwasawa, and Nagahama;
unpublished data). An aquatic habitat appears
unnecessary for the PRL-stimulated growth of tail
fin, but assists its rapid growth. If a high level of
PRL is seen in animals during the terrestrial phase,
a little growth of tail fin must occur at that time
independently of entering the water. Accordingly,
the absence of PRL-stimulated increase in tail
height of winter-dormant male H. nigrescens kept
in a terrestrial habitat [5] may be due to the
comparison of a little increase in tail height of
these animals with no increase in that of control
ones; the comparison between tail heights of the
same individual was made in the present study.
Regardless of the type of treatment (i.e., saline
or PRL), all animals housed in a fully aquatic
1095
ATiL® ° AS ° TS
B e e
Cc e
D e
E e
F e
G e
A e e AP e e TP
B e e e e
Cc e e e ee
D e e ee
E e ee ee
[= ee e ee e
Gi e e e
fh ft
CPaTa Pree 8 es © oe fw oe we
DAYS
Fic. 2. The frequency of molting (solid circle) in groups
AS, AP, TS, and TP (see the caption of Table 1) at
8°C. A-G: individual numbers.
habitat molted at least once during the ex-
perimental period (Fig. 2). Regardless of habitat
(i.e., terrestrial or aquatic), PRL induced frequent
molting. Smooth molting occurred in group AP,
but not in group TP. Vellano ef al. [1] reported
that in thyroidectomized T. cristatus PRL induced
molting. Dent et al. [7] stated for N. viridescens
that PRL alone did not induce molting but might
facilitate molting when introduced with thyroid
hormone. Probably PRL, apart from thyroxine,
stimulates molting in H. nigrescens. Because the
present July materials were seasonally immature
[8], concentrations of the hormones associated
with aquatic reproduction (e.g., PRL [10]) would
be expected to be low. Despite this expectation,
even under low-temperature conditions, molting
was speeded up in all saline-injected males housed
in a fully aquatic habitat in comparison with those
in a terrestrial one. On the other hand, most of
those saline-injected, mature male H. nigrescens
that temporarily submerge in water possess the
surface layer (cornified epidermis), indicating that
temporary submergence is not enough to induce
molting [5]. All of these observations lead us to
postulate that a fully aquatic environment induces
the secretion of PRL in this species.
ACKNOWLEDGMENTS
We would particularly like to thank Dr. Y. G.
Watanabe of Niigata University and two anonymous
reviewers for critically reading the manuscript. This
1096 M. Hasumr AND H. IwasAwa
study was supported in part by a Grant-in-Aid for
Scientific Research to HI from the Ministry of Educa- 6
tion, Science, and Culture of Japan.
48: in press.
Fox, H. (1986) In “Biology of the Integument. 2.
Vertebrates”. Ed. by J. Bereiter-Hahn, A. G.
Matoltsy and K. S. Richards, Springer-Verlag,
Berlin, pp. 78-110.
REFERENCES 7 Dent, J.N., Eng, L. A. and Forbes, M. S. (1973) J.
Vellano, C., Mazzi, V. and Sacerdote, M. (1970) Exp. Zool., 184: 369-382.
Gen. Comp. Endocrinol., 14: 535-541. 8 Hasumi, M., Iwasawa, H. and Nagahama, Y. (1990)
Singhas, C. A. and Dent, J. N. (1975) Gen. Comp. COVE SE UIST.
Endocrinol., 26: 382-393. 9 Hasumi, M. and Iwasawa, H. (1990) J. Herpetol.,
Kikuyama, S., Seshimo, H., Shirama, K., Kato, T. Be UBS.
and Noumura, T. (1986) Zool. Sci., 3: 131-138. 10 Mazzi, V. and Vellano, C. (1987) In “Hormones
Hasumi, M. and Iwasawa, H. (1987) Zool. Sci., 4:
159-166.
Hasumi, M. and Iwasawa, H. (1992) Herpetologica,
and Reproduction in Fishes, Amphibians, and Rep-
tiles”. Ed. by D. O. Norris and R. E. Jones, Plenum
Press, New York, pp. 87-115.
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ILGSOGCLICTUSIRG vrais crite ices iolsrsvarniacins oat sanabev aed 1037
Hasumi, M. and H. Iwasawa: Dependence of
prolactin-stimulated tail fin growth and molt-
ing on water in male salamanders (Hynobius
nigrescens) (COMMUNICATION) .... 1093
Kezuka, H., M. ligo, K. Furukawa, K. Aida
and I. Hanyu: Effects of photoperiod,
pinealectomy and opthalmectomy on circu-
lating melatonin rhythms in the goldfish,
GaKGSStUSVQUIGIUS. <0. eben eect eee ee 1047
Peter, V.S. and O. V. Oommen: Intermedi-
ary metabolism in _ castrated/thyroid-
ectomized Calotes versicolor: Regulation
by thyroxine and testosterone .......... 1055
Arakawa, E., T. Kaneko, K. Tsukamoto and
T. Hirano: Immunocytochemical detection
of prolactin and growth hormone cells in the
pituitary during early development of the
Japanese eel, Anguilla japonica ........ 1061
Morphology
Ishikawa, Y.: Innervation of the caudal-fin
muscles in the teleost fish, medaka (Oryzias
URNA TOS\\ see eercrn anc cene ol ano oe ene 1067
ZOOLOGICAL SCIENCE
VOLUME 9 NUMBER 5
CONTENTS
REVIEWS
Takahashi, S.:
ment of somatotrophs and mammotrophs in
Heterogeneity and develop-
thestat? (.c258 ee Rot ee eee 901
Gerencser, G. A. and B. Zelezna: Chloride
pumps in biological membranes .......... 925
ORIGINAL PAPERS
Physiology
Azuma, K., N. Iwasaki, M. Azuma, T. Shino-
HPLC Analysis of
retinoids extracted from the planarian,
zawa and T. Suzuki:
Dugesia japonica
Fukuta, S., T. Ikata and I. Miura: Effect of
disuse on muscle energy metabolism studied
by in vivo 31-phosphorus magnetic reso-
nance spectroscopy
Ootsubo, T. and M. Sakai: Initiation of sper-
matophore protrusion behavior in the male
cricket Gryllus bimaculatus DeGeer
Cell Biology
Lee, Y. H. and C. E. Lee: Uktrastructure of
spermatozoa and spermatogenesis in Nepo-
morpha (Insecta: Heteroptera) with special
reference to phylogeny
Immunology
Rinkevich, B. and Y. Saito:
the colonial
Self-nonself rec-
ognition in protochordate
Botryllus schlosseri from Mutsu bay, Japan
Rinkevich, B., M. Shapira, I. L. Weissman and
Y. Saito: Allogeneic responses between
three remote populations of the cosmopoli-
tan ascidian Botryllus schlosseri
Saad, A. H.: Sexual development of im-
os
OCTOBER 1992
munocompetence in the toad, Bufo regularis
(COMMUNICATION) .......5aee 1081
Fabry, H. and J. L. Hedrick: Antibody pro- —
duction in the goat: Immunokinetics and
epitope specificity using a glycoprotein im- k
munogen
Biochemistry
Asami, K.: Appearance of a nuclear histone
H1 kinase at the start of DNA syne of
regenerating rat liver
acc a
Developmental Biology
Kobayashi, H. and T. Hishida: Electron-
microscopic observation of an ectopic PGt
like cell in the teleost Oryzias latipes (COM-
MUNICATION)
ee eo ee of el she Se
Reproductive Biology
T., H. Yamanaka, K. Suzuki, K
Nakajima, K. Kanatani, M. Kimura and N.
Otaki: Immunohistochemical demonstra-
tion of metallothionein in the rat rid iS
and spermatic cord
Kubokawa, K., S. Ishii, K. Tanabe, K. Saitou
and H. Tae
feces of giant pandas
Suzuki,
Analysis of sex steroids 11
Endocrinology
Paolucci, M., M. M. Di Fiore and G. Ciarcia:
Oviduct 17-estradiol receptor in the fen
lizard, Podarcis s. sicula, during the sexual
cycle: Relation to plasma 17/-estradiol
centration and its binding proteins
Saad, A. H., M. H. Mansour, M. E. Yazjia
N. Badir:
trols humoral immunity in the lizard, Chal-
Endogenous testosterone
(Contents continued on inside back cover)
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REVIEW
Recent Progress in Comparative Neuroimmunology
BERTA SCHARRER
Department of Anatomy and Structural Biology, Albert Einstein
College of Medicine, Bronx, New York 10461, USA
INTRODUCTION
During the past decade, broadly based compara-
tive studies on cell-mediated immunoregulatory
processes have revealed remarkable parallelisms
between vertebrates and higher invertebrates (see
Scharrer [1]). One distinctive feature these two
groups have in common is the use of the same or
closely related chemical messenger substances in
internal immunoregulatory processes as well as the
bidirectional exchange of information between the
immune system and the neuroendocrine system
[2]. Neuropeptides, formerly considered to be
confined to the neuroendocrine apparatus, are
now known to be produced by and released from
immune cells. By the same token, cytokines, i.e.,
regulatory molecules characteristic of the immune
system, have been shown to be manufactured also
by neural elements, including astrocytes. Informa-
tion on the identities of these signal molecules in
invertebrates as well as vertebrates has been
obtained by use of biochemical and immunocy-
tochemical methods, and by in vitro and in vivo
tests on the effects of various exogenous analogs.
The immunoactive cells of the invertebrates that
have been primarily used in these studies, the
molluse Mytilus edulis and the insect Leucophaea
maderae, are subpopulations of the animals’ hemo-
cytes. They share a number of properties with cells
of the mammalian granulocyte-monocyte-macro-
phage lineage. Evidence for the existence of
different subsets of invertebrate immunocytes was
obtained by the observation of differences be-
Received August 10, 1992
tween the modulatory effects of opioid peptides
and those of certain other ligands [3]. Like their
counterparts in vertebrates, these cells respond to
immunomodulatory stimuli by characteristic con-
formational changes indicative of cellular activity.
For these light microscopic observations immuno-
cytes were incubated together with various ligands
at 37°C for human, and at 23°C for Mytilus and
Leucophaea. Either a Zeiss Axiophot Microscope
and the Zeiss Videoplan/Vidas Image Analysis
System [4] or the system designed by American
Innovision, Inc. were used. Changes in cellular
conformation were determined by measurements
of cellular area and perimeter, mathematically
expressed by use of the Zeiss Form-Factor (FF)
forumula [4]. Additional factors, introduced by
Schon et al. [5], permitted a distinction between
types of cellular change, i.e., those obtained in
different animal species by the same signal mole-
cules and those effected by different substances in
the same species.
Neuropeptides
The search for multiple functional roles of
neuropeptides in signaling within as well as outside
of the nervous system has taken a number of
unexpected turns since their discovery in special
neurosecretory neurons some 70 years ago. Cur-
rently available evidence indicates that members
of this large family of compounds, especially
opioid peptides, may have dose-dependent stim-
ulatory as well as suppressive effects on various
immunoregulatory processes. The substances
acting in these capacities are furnished by im-
munoactive cells as well as cells of the neuroendo-
1098 B. SCHARRER
crine system.
The capacity of invertebrate immunocytes to
produce and release a Met-enkephalin-like mate-
rial was demonstrated by high pressure liquid
chromatogaphy and radioimmunoassay. This ma-
terial was found to be present in cell-free
hemolymph as well as hemocytes of Mytilus [4].
Evidence for its presence in Leucophaea hemo-
lymph is more tentative (see Scharrer et al. [6]).
Met- and Leu-enkephalin was also identified in the
pedal ganglia of Mytilus [7].
Immunocytochemical methods have revealed
substances antigenically related to Met-enkephalin
and numerous other neuropeptides in the neuroen-
docrine system of Leucophaea and other insects
(see Scharrer et al. [6]).
1. Stimulatory activities
Immunocytes of invertebrates and vertebrates
respond to stimulation with appropriate concentra-
tions of opioid and other peptides by increased
adherence to albumin-coated slides and the forma-
tion of clumps. Prior to becoming mobile, these
cells flatten and change their structure from round-
ed to ameboid (Mytilus) or elongated (Leuco-
phaea, human). The addition of the opioid antag-
onist naloxone to the preparation, concurrent with
the cells’ exposure to exogenous opioids, blocks
their activation. Activated immunocytes of Myti-
lus have been observed to move in the direction of
cell clusters of the same type [4].
Moreover, in vivo experiments with this mollusc
or mammalian laboratory animals showed that
chemotactic movements of immunoactive cells are
guided by concentration gradients of certain spe-
cific recognition factors. Thus an immune-type
response, experimentally induced in Myzilus by the
severance of a nerve, resulted in the migration of
immunocytes and their gradual accumulation in
the lesioned area. After the injection of DAMA
(D-Ala*-Met®-enkephalin) into an intact animal,
but not that of a number of control substance, the
cells accumulated at the site of injection [4].
An important feature observed by Stefano er al.
[8] was that the stimulatory effect of Met-
enkephalin on cellular conformation and locomo-
tor activity is significantly higher than that of
related neuropeptides or nonpeptide substances.
This information was initially obtained by tests
with the synthetic analog of Met-enkephalin,
DAMA, which is resistant to the effect of the
naturally occurring neutral endopeptidase 24.11
(NEP). The same high potency values (107 '' M)
were accomplished by the administration of Met-
enkephalin or of the heptapeptide Met-
enkephalin-Arg®-Phe’ with the addition of the
special enzyme inhibitor phosphoramidon [9-11].
In eight additional drugs examined, including the
closely related Leu-enkephalin, the required con-
centration for comparable stimulatory effects was
in the area of 10~° M. Beta-endorphin was active
at 107 !°M [8].
The search for stereoselective mechanisms
mediating the immunostimulatory effects of
opioids and nonopioid substances has provided
pharmacological and binding evidence for the pre-
sence of receptors on immunoresponsive cells.
The results of Scatchard analysis indicate that
these binding sites on human granulocytes and
Mytilus immunocytes are monophasic, stereospe-
cific and of high efficiency. In both vertebtrate and
invertebrate tests the complexity of ligand-
receptor binding observed turned out to be greater
than anticipated. Multiple opioid receptors, in-
cluding delta-, mu-, kappa-, and presumably epsi-
lon-receptors are preferentially used by different
messenger molecules performing selective func-
tions [8].
The high potency of Met-enkephalin demon-
strated in in vitro tests with Mytilus and human
granulocytes suggests that it plays a distinctive role
in immunoregulation. On the basis of mammalian
in vivo experiments and of preliminary clinical
tests, Jancovi¢ et al. [12] called attention to the
potential value of Met-enkephalin in the treatment
of certain immune diseases. The proposal made by
Stefano ef al. [8] that, in its immunoregulatory
role, Met-enkephalin interacts with a special sub-
type of opioid receptor (delta>) has been substanti-
ated by recent experiments with the equally potent
opioid (D-Ala*) deltorphin I [13]. Preincubation
of human granulocyte membranes with DALCE (a
select detla-opioid antagonist) revealed a binding
capacity of this opioid, as well as that of DAMA,
distinctly different from that of other opioids.
In comparing the modulatory effects of various
Comparative Neuroimmunology
opioids on certain growth processes, Zagon et al.
[14] likewise found Met-enkephalin to be more
potent than the rest. This pentapeptide had a
strong inhibitory effect on the growth of neuro-
blastoma cells in tissue culture. The special recep-
tor proposed to be selectively involved was found
in abundance in the cerebellum of infants and in a
human brain tumor, but not in the cerebellum of
normal adults.
2. Immunosuppressive effects
In contrast to most of the neuropeptides tested
thus far two, derived from pro-opiomelanocortin,
i.e., ACTH and MSH, are immunosuppressive.
When tested in appropriate concentrations, they
have been shown to inactivate immune cells that
are either spontaneously active (5-10% in un-
treated preparations) or have been experimentally
activated by other signal molecules known to have
a stimulatory effect [11]. Part of this activity of
ACTH seems to be indirect, i.e., by its conversion
to MSH under the influence of the special en-
dopeptidase (NEP) mentioned earlier. These
observations have been made in experiments with
vertebrate and invertebrate immune cells.
Information on additional immunoregulatory
effects brought about by various neuropeptides is
primarily based on mammalian studies. Such
effects include antibody production, histamine re-
lease from mast cells, cytokine activity, vasodila-
tion in inflammatory area, proliferation of lympho-
cytes, and phagocytotic activity.
Cytokines
In the search for basic immunoregulatory princi-
ples shared by invertebrates and vertebrates, the
results of pilot studies on the presence and activity
of cytokines in molluscs and echinoderms promise
to be as rewarding as those on neuropeptides. The
hemolymph of Mytilus was found to contain im-
munoactive interleukins (IL-1 and IL-6) as well as
tumor necrosis factor (TNF-a). Its immunocytes
respond to the respective mammalian cytokines in
vitro and in vivo in a manner comparable to that of
human granulocytes [15]. Like in mammals, IL-1
appears to participate in the internal regulation of
the immune system of Mytilus in part indirectly,
i.e., by its stimulatory effect on the formation of
1099
TNF. Moreover, IL-1 and TNF were demon-
strated to be present in the hemolymph of
Leucophaea (YT. K. Hughes, Jr., Personal com-
munication).
A molecule with the biochemical and biological
characteristics of mammalian interleukin 1 has also
been reported to be produced by an echinoderm,
Asterias forbesi [16].
Mode of operation of immunoregulatory substances
Neuropeptides, cytokines and additional factors
(including special enzymes) interact to form an
efficient immunoregulatory network. Depending
on their concentration and additional determining
factors present, certain messenger molecules may
be either immunostimulatory or -inhibitory. In
addition to its immunosuppressive function, men-
tioned earlier, ACTH may stimulate the activity of
B-lymphocytes and the phagocytotic capacity of
molluscan immunocytes [17]. Its own production,
as well as that of endorphins, by immune cells can
be induced by another neuropeptide, corticotropin
releasing hormone [18]. Conversely, the synthetic
opioid DAMA has been shown to stimulate the
formation of an IL-1-like molecule in human and
Mytilus immune cells [11].
One way in which the degree of cellular activa-
tion is kept within appropriate limits is by enzy-
matic degradation of some of the signal molecules
involved [10]. The observation that the local
concentration of Met-enkephalin (and of the re-
lated heptapeptide Met-enkephalin-Arg®-Phe’) is
downregulated by NEP also applies to the inverte-
brate Mytilus [11]. Tests with two major com-
pounds resulting from the hydrolysis of the hep-
tapeptide showed that they antagonize its effect,
presumably by competing for part of the same
receptor sites as those used by the heptapeptide.
A balancing mechanism is thus provided by the
enzymatic generation of antagonistic fragments
from the agonist’s molecule.
Interference with the normal operation of the
immunoregulatory network may occur in various
ways and may have important biomedical conse-
quences. In schistosomiasis, immunosuppression,
attributable to the release by an invertebrate para-
site of immunosuppressive signal molecules re-
sembling those of the host, appears to influence
1100
the course of the disease [19]. Similarly, the
survival of the human immunodeficiency virus in
patients seems to be supported by its ability to
stimulate the production of ACTH by the host
[20].
An involvement of neuropeptides in the re-
sponse to stressful conditions has been demon-
strated not only in vertebrates, but also in the
mollusc Mytilus [21]. The animal’s immune/de-
fense system can be alerted, for example, by
mechanical interference with the valves of the
in-current syphon. This leads to a significant rise in
the number of “activated” immunocytes, pre-
sumed to be due to the release of an endogenous
opioid-like material from the brain.
In conclusion, the rewards gained from a broad-
ly based comparative approach to the study of
neuroimmunological phenomena reach beyond the
elucidation of commonalities between vertebrates
and invertebrates. They provide information on
the evolutionary history of a basic biological phe-
nomenon and open new vistas in its exploration.
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3 Hughes, T. K., Jr., Smith, E. M., Barnett, J. A.,
Charles, R., and Stefano, G. B. (1991) Cell Tissue
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4 Stefano, G. B., Leung, M. K., Zhao, X. and
Scharrer, B. (1989) Proc. Natl. Acad. Sci. USA, 86:
626-630.
5 Schon, J. C., Torre-Bueno, J. and Stefano, G. B.
(1991) Adv. Neuroimmunol., 1: 252-259.
6 Scharrer, B., Stefano, G. B. and Leung, M. K.
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ZOOLOGICAL SCIENCE 9: 1101-1111 (1992)
REVIEW
Rhythmic Processes in Lower Vertebrate Embryogenesis and
Their Role for Developmental Control
Yuriy N. GORODILOV
Biological Institute of St. Petersburgh University, Oranienbaum
Chaussee 2, Stary Peterhof, St. Petersburgh 198904, Russia
ABSTRACT—A great number of various rhythmic processes (biochemical, biophysical, cytological)
during an animal embryogenesis is described up to now. Many of them are revealed in the course of
cleavage. The duration of a single cycle of these processes is usually equal to the duration of a single
cleavage division (Zo).
The somitogenesis is demonstrated the feature of rhythmic process too. We studied thoroughly
increase of somites in many fish species, particularly in slow-developing salmon fishes allowing most
exact measurements. It was shown that the formation of somites, next one after another, occurred (at
constant conditions) strictly rhythmically with the same interval (ts). The last is specific for each
species. It turned out that in embryos of the same species at the resemble temperature interval z, was
equal to. These data permit to suppose that both embryonic processes are governed by the same
intrinsic rhythm, peculiar to all cells of the embryo. This general conclusion is sustained by the data
showing that in some salmon hybrids when the reorganization of the rhythms occurred a similar shift
in the frequency of the ones for both processes was observed. It is possible that the autonomous
changes in the competence of amphibian ectoderm for induction are also governed by the same
endogenous rhythm.
The endogenous rhythm described could be responsible for switching on/off various specific
programmes of development, and also for the coordination of numerous developmental events of
embryogenesis. It is supposed that various programmes switched on/off by the same rhythmic signal
© 1992 Zoological Society of Japan
can be performed independently.
INTRODUCTION
In adult organism, temporal coordination of
various aspects of life processes is usually accom-
plished by means of periodic external phenomena
like day and night, seasons, etc. Do organisms
have any internal time control mechanism during
the embryogenesis?
This period is remarkable for relative concise
and extraordinary eventfull processes of dif-
ferentiation and morphogenesis. One is tempted
to ask how the activity of enormous numbers of
genes is organized, not forgetting that gene ex-
pression is only the first step in the multistage
Received August 14, 1992
process when the realization of genes in characters
occurs.
It has been generally believed that the develop-
ment of an organism is a chain of consecutive
stages: “Development seems to occur in a stepwise
fashion, the completion of each step setting the
stage for the next” [1]. If this is the case, the
duration of a process depends on environmental
factors (temperature, oxygen, pH, etc.), that’s say
the time is derivative of those ones, and does not
play the role of the primary factor of development.
The proponents of this point of view believe that
the duration of different embryonic processes is
usually not stirctly constant exploring it by the
imperfections of control of each process. This
hypothesis provides the basis for an opinion that
1102
the development of embryos from one batch is not
simultaneous, as it is shown by non-simultaneous
hatching of embryos.
According to Crisp [2] the duration of
embryogenesis is one batch estimated at this stage
can differ by 20%. But hatching depends on a set
of environmental factors and can occur at different
stages of development [3].
The point of view that embryogenesis is a pro-
cess with relatively variable time limits is beginning
to change actually. This is the consequence of
more appropriate methods using allowing to esti-
mate quantitative aspects of embryonic develop-
ment and of a series of direct experiments which
are studying the time factor in the process of
development. Several rhythmic processes in
embryos indicate the existence of some embryonic
time control mechanism or “biological clock” [4].
Several of these rhythmic processes showing
similarities in particlar aspects are described
below.
RHYTHMIC PROCESSES DURING CLEAVAGE
Cleavage is the earliest period of the
embryogenesis, but several rhythmic processes,
including cell divisions, are observed already at
this stage. These cleavage cell divisions occur
synchronously in 10-12 cycles [5—6] with approxi-
mately equal time interval between similar phases
of mitosis. This interval was designated To [7].
As it has been recently shown, rhythmic changes
also occur during this period in biochemical and
biophysical parameters. The most interesting ones
are periodic changes of rigidity in the superficial
layer of the egg cytoplasm [8], flattening-rounding
of the egg and surface contraction waves and other
changes [9-11] (for review of these and other
changes see [12-14]). These processes occur at
time intervals, which are approximately equal in
duration to the cell cycle duration in cleavage.
They occur both in intact and enucleated eggs and
in egg fragments not containing nucleus. Besides,
it has been shown that the changes of an electric
activity of embryonic membranes occur also with a
period equal to the cell cycle [15-16].
Y. N. GoropiLov
THE TIME CONTROL IN EMBRYOS AND
SINGLE EMBRYONIC CELLS AT STAGES
FOLLOWING CLEAVAGE
How is the time controlled in embryo cells after
cell divisions are desynchronized during blastula-
tion? The following answer on this question is
possible: since the cyclic changes of some indices
remain after the suppression of cleavage [12-14] it
could be supposed that the same occurs when the
cell cycle reorganization and the loss of rhythm by
cell divisions takes place in intact embryos, namely
rhythm of another processes retain. Some data
may be presented, which prove the existence of
temporal control in the embryos and their cells
following the cleavage.
It has been ascertained in series of experiments
that the beginning of blastulation and gastrulation
in different groups of animals does not depend on
the nuclear-cytoplasm ratio or on the number of
divisions, but on the duration of development [17-
19]. It has been shown also that the time of
realization of different processes in either anlages
of embryo programmed in the cells and the sche-
dule of their development (with one moment or
another) occurs independently from neighbouring
anlages. For example, at the fusing of starfish
embryos at different stages of development, in
spite of the formation of highly permeable contacts
between them, every embryo starts the gastrula-
tion by own schedule [20]. The attempts to change
the time of beginning of gastrulation using prema-
ture processing of embryos with the mesoderm-
inducing factors have been unsuccessful [21]. The
latter authors think that each cell in the early
embryo contains a copy of the schedule controlling
the correct initiation of developmental events,
whether or not that cell has been instructed to
participate in each event. Grainger and Gurdon
[22] have shown that the loss of ability of
mesodermal induction in ectodermal cells does not
depend on cellular interactions and cell divisions:
isolated cells lost their competence simultaneously
with intact embryos. Further it has been demon-
strated [23] that in Xenopus the cultured ectoderm
undergoes the set of successive changes: first it is
competent to form mesoderm, then neural tissue
and finally placodal tissue. The authors conclude
Rhythmic Processes in Embryogenesis
that the competence of ectoderm changes over
developmental time, and that no tissues interac-
tions are required for these changes in compe-
tence.
It seems that the time control mechanism keeps
at later stages and has certain significance for
morphological differentiation of cells already in
the separate anlages of organs. So, the ability of
chick embryo limb bud cells to differentiate into
distal or proximal region of the wing is dependent
on the duration of a period which the cells spend in
the growth zone [24].
It seems that switching on/off the synthesis of
several specific enzymes and chemical substances is
also controlled by an intracellular embryo clock.
A tissue-specific enzyme acetylcholinesterase
(ACh E) is found in muscle cells of Ascidia
embryos [25]. If at the stage of 32 blastomeres
ascidian eggs were treated with cytochalasin
(which inhibits cytokinesis, but does not suppress
nuclear divisions) or colkhicine (which inhibits
nuclear divisions) in these seemingly undeveloping
embryos ACh E activity appeared at the same
moment as in intact embryos [12, 25, 27]. Caplan
[27] also pointed to the existence of a biological
clock in the synthesis the specific progeoglycans in
chondrocytes both in embryonic organs and iso-
lated cells.
SOMITHOGENESIS AS A RHYTHMIC
PROCESS
The axial zone in vertebrates is subject to seg-
mentation the first step of which is separation of
parachordal mesodermal layers in rostro-caudal
direction and formation of somites. They are
formed in pairs, by one on each side of chorda.
Their total number ranges from several tens to
several hundreds of pairs. As has been shown
earlier, the formation of somites is a relatively
rhythmic process [28-30].
We studied somitogenesis in several species of
teleosts at different temperatures. Teleosts
embryos represent a convenient model for live
observations since they are transparent.
In the laboratory the fertilized eggs of different
fish species placed into special apparatus for in-
cubation of salmon (and also other) fishes. During
1103
incubation normal conditions of spawn develop-
ment (oxygen concentration, flowing water, con-
stant temperature (+0.1°C) maintained carefully.
Within the optimal temperatures (for salmon eggs
2-10°C) our incubators are a success to provide the
survival of 95-98% embryos up to hatching [31,
33].
In the course of somitogenesis we took out the
samples of eggs for counting of somite pairs from
the same batch from time to time. For the sake of
this the chorions were removed microsurgically in
a twofold Holtfreter solution, the yolk was drawn
off and embryos were carried by pipette on the
preparation glass. Then we counted the number of
somite pairs in every embryo under microscope
(enhance 7X8). The production of 5-10 living
embryo preparations and the counting of somites
in them usually took no more than 30-50 min.
Numbers of somite pairs in each sample under
investigation were averaged and plotted on the
grafic of increase of somite pair sum in dependence
on the incubation duration.
In a series of observations of embryos from one
batch it was found that at constant temperature the
total number of somites is a linear function of
incubation time (Fig. 1; see also [3, 31-34]). This
tule is retraced from the laying of first somite pair
up to 35 pairs in perca [34], 57 pairs in pike [32], 60
pairs in Atlantic salmon [34] and 64-65 pairs in
chum (Fig. 1). It is only the last several pairs in
each species that are formed slower.
The fact that dependence of the somite pair
number upon the incubation time has the strictly
linear character means that formation of somites,
one after another, occurs with the one and same
temporal interval. This circumstance allows to
calculate a given interval as the time of formation
of one somite pair Zs:
Ca Ei
CSSamasen 1S
n2—Ny
were 7, and rf are the moments when somite pairs
nN, and np respectively started to form. The more is
the difference nj—n,, the more exact estimation of
ts can be obtained.
It is necessary to point out the high developmen-
tal synchroneity that is observed in embryos of all
species and all batches during the period of somi-
1104
Somite
pairs
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ee
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fotidits 16 eooltermeda Ip gone
0 200 400 600 hour
Fic. 1. Increase of the number of somites in chum
Oncorhynchus keta Walbaum during axial seg-
mentation depending on incubation time at various
temperature regimes: 11.3°C (1), 6.0°C (2), 3.7°C
(3). Time since the beginning of somitogenesis
observations (hours) is marked on the horizontal
axis, the number of somite pairs is marked on
vertical axis.
togenesis. The synchroneity keeps at the same
level at the beginning and at the termination of
somitogenesis period. For example, we reveal the
data of two plots in Table 1 received from the
Atlantic salmon embryos from Kola river popula-
tion at temperature of 0.4°C. The average value of
somite pair number of 12.8 for the first sample had
been received from studying of 7 embryos: 6 ones
among them had 13 pairs each and one had 12
pairs. After 840 hr (35 days) we have counted
somites of 6 embryos from another sample of the
same batch. They were 56, 57, 57, 57, 57, and 57
(56.8 in average). We observed the similar level of
synchroneity in other instances that can be seen
from the meanings of statistic deviation from aver-
age numbers of somite pairs (Table 1 and 2).
Y. N. GoropILov
Thus the somitogenesis at constant conditions
appears to be the strictly rhythmic process which is
controlled with extraordinary high precision. The
data of Table 1 and 2 proves that rg is a constant
temporal characteristic for all embryos of a given
species independently on the time and place of
spawn sampling. Deviation of ts in parallel sam-
ples is usually less than 1% or in some cases 0.1%,
that is the absolutely unusual phenomenon for
biological processes.
If embryonic development is controlled by any
cyclic process, the last one should have the same
characteristics in all embryos of a species, but
these characteristics can be different in various
species, as we see in somitogenesis. For example,
in chum and Atlantic salmon at 6.0°C the duration
of each cycle of somitogenesis is 368 and 401 min
respectively (Table 1 and 2), in pike at 9.9-10.0°C
it is approximately twice shorter than in chum
(Table 2). It would be said that the embryos of
each species have their own time counting off that
determine specine-specific “own time” of develop-
ment.
THE RHYTHMS OF PERIODIC PROCESSES
IN TELEOSTS AND AMPHIBIANS AND
THEIR INTERCONNECTIONS
If we compare the duration of the cycles of
cleavage divisions to and that of somitogenesis Ts
it is possible to see that at the particular tempera-
ture they are similar. We can compare two curves
of dependence of these parameters from the in-
cubation temperatures received for Atlantic sal-
mon (Fig. 2). Similar proportions between 7 and
Ts are observed in other species. It can be easily
traced from the comparison of to and Ts values in
species having different “own time”. As it can be
seen from Table 3, species with higher cleavage
rate (rainbow trout) also have higher somitogene-
sis rate and vice versa (Atlantic salmon).
In Fig. 3 the duration of the first five cleavage
divisions determined cytologically [6] and that of
the formation of any five somites at the same
temperature (4.8 °C) is compared. Here it can be
clearly seen that the cleavage rhythm that was lost
during the subsequent asynchronous cell divisions
seems to reappear during somitogenesis after some
Rhythmic Processes in Embryogenesis
TABLE 1.
1105
Determination of time of somite formation (zs) in Atlantic salmon (Salmo salar L.) embryos
from different populations during egg incubation at several constant temperature
No. of somites
ae tare gg ation ca of Ree No. of embryos pouites a ae
CO) vey) euser (hr) onset end pene (min)
Kola! 5/XI 840 aoe ee 44.0 1146
a Neva? 28/XII 818 a eee 42.8 1147
Kola 9/XI 580 cian ee 48.2 2
i Neva 6/XII 581 St see 47.9 720
Kola 4/X1 291 are ie 43.7 400
6.0 Neva 17/X1 335 ee mene 49.9 403
Salatza? 14/X1 306 coat’ eres G0 309
Kola 28/X 164 —_ =e 43.6 225
9.7 Neva 11/XI 164 ae Se 43.6 226
Salatza 9/XI 163 ae a 43.8 222
' Barents sea basin (69° N.L.)
? Baltic sea basin (60° N.L.)
3 Baltic sea basin (57° N.L.)
hundreds hours.
Incomplete coincidence of to and zg in investi-
gated fish species at several temperatures may be
stipulated by different factors. Firstly, the dura-
tion of different cleavage cycles, evaluated either
by cytokinesis and mitotic index, are not always
coincided [37]. Secondly, the activity of inner
oscillator is registered only through external pro-
cesses that can not fully adequately reflect this
inner rhythm.
It should be noted that in all fish species studied
T and zs give the ratio of 1:1, but this ratio is
different in common frog Rana temporaria: at
15°C the duration of cleavage cell cycle in frog is 70
min [38] and of the formation of one somite pair is
140 min [39], i.e. the zo/zs ratio is 1:2. Conse-
quently, for the similar event in reality in one case
(fishes) the time equal to Zo is spent, in other case
(amphibia) is twice as much. The existence of
divisible ratio seems to be caused by the rhythmic
processes under discussion submitted to endogenic
rhythm generated in embryo with the main period
approximately equal to zo. In dependence on
complexity and specificy of the process, it manages
in one, two and more periods of endogenic
rhythm. It can be supposed that the duration of
morphogenesis of other organs and that of the
period of postulated endogenic rhythm are relating
as divisible numbers.
COMPARABLE TRENDS OF CHANGES
OF EMBRYONIC RHYTHMS IN
INTERSPECIES HYBRIDS
We studied the embryonic rhythms in hybrids
between the Pacific salmon species: pink
1106
TABLE 2.
Y. N. GoropDILov
Determination of time of somite formation (zs) in chum (Oncorhynchus keta Walbaum) embryos
from different populations and pike (Esox lucius L.) embryos from different accessions during egg
incubation at several constant temperatures
No. of somites
5 P lati 5 ,
Rest ce species cen) Date Dalat of | No:ofembyos’ (Sone eae
(CS) of accession ONSe! (hr) onset end (min)
chum —_Naiba! 11/X-81 336 aneee ae 54.5 370
6.0 chum Zavetinka? _14/X-86 319 ihe a 51.9 369
gum Tae 26/X-86 260 ag a 42.7 364
chum Zavetinka 14/X-86 122 Beste eel 36.3 201
i dima | Then 26/X-86 118 eee neh 34.7 204
pike 1 29/1V 90.5 a ee 48.3 112
ue pike 2 2/V 78.0 o2et2 2 43.3 108
pike 1 2/V 54.8 —— ae 40.6 81
oe pike 2 2/V 57.4 ages sent 43.7 79
Ww N =
(Onchorhynchus gorbuscha) and masu or cherry
salmon (O. masu). It was found out that masu has
a higher rate of somitogenesis than pink at all
temperatures (Table 4). The comparison of Ts
parent species and hybrids (Table 4) showed that
the rate of somitogenesis in hybrids deviated from
both parents but was closer to that of male parents.
In 1989-1990 we could not obtained masu
females, so we compared Tg only in pink and in the
hybrid where masu was a male parent. At all four
temperature regimes tz in the hybrid was shorter
than in the maternal species.
If cleavage and somitogenesis rhythms have
anything in common, the shift in rhythm of one
process in a hybrid should be followed by an
adequate shift in the other. That was the reason
why we studied cleavage rate at one temperature
(10.1°C) in masu, pink and their hybrids (Table 5).
In masu cleavage, as well as the somitogenesis,
The river on the Western coast of island Sakhalin, entering the Sea of Okhotsk (47.5° N.L.).
The river on the eastern coast of island Sakhalin, entering the stream of Tartary (47 N.L.).
The river in the north of island Sakhalin, entering the Sea of Okhotsk (52° N.L.).
is completed faster than in pink, that additionally
confirm the existence of correlation between the
rates of these processes in the same species. The
early three cleavage divisions (II-IV) in masu eggs
take 83 min less than in pink ones, i.e. t) in masu
at 27-28 min shorter than in pink. The ro duration
determined as the average time of three cleavage
cycles: (II-IV) in all variants practically coincided
with to, measured as one cycle duration between
cytokinesis of the first and the second divisions.
The measurement of zt» in hybrids demonstrates
pronounced the shift in the development rate of
maternal eggs owing to the influence of father
genome: in pink eggs the cleavage rate is enhanced
when they were fertilized by the masu males. On
the contrary, in masu eggs fertilized by the pink
males the cleavage rate became slower (Table 5).
Consequently the rhythms of cleavage and somi-
togenesis in hybrid embryos change by the similar
Rhythmic Processes in Embryogenesis 1107
0 2 4 6 8 10 12 14
"C
Fic. 2. Duration of cell cycles during cleavage (79) and
of the formation of one somite pair during linear
somitogenesis (zs) in Atlantic salmon embryos de-
pending on the temperature of incubation. Horizon-
tal axis, temperature. Vertical axis, duration.
way: if one process is accelerated, the other pro-
cess 1s accelerated too, and vice versa.
It should be noticed that the inheritance of the
cleavage rate in hybrids of considered variant is
evidently influenced by the male parent. It breaks
up the old presentation that the cleavage rate is
dependent on the egg cytoplasm only (for review
see [13]). Thus the evident similarity in the
rhythms of cleavage and somitogenesis, taking
place at the different periods of embryogenesis,
definitely proves the existence of common biolog-
ical time in embryos for these periods, and perhaps
for other periods of embryo development as well.
AGE-RELEVANT CHANGES IN COMPETENCE
Competence is defined as the ability of embryo
tissues to respond to an inductive signal that
activates particular developmental programmes
[41-42]. It has been found that the competence of
the embryo tissues changes with time, following a
strict programme.
Holtfreter [43] observed the changes in the
ability of isolated ectoderm of the early gastrula
cultivated for different periods in a salt medium to
form different rudiments. It is important that
these changes occur in a stepwise and autonomous-
ly. This was confirmed by recent studies.
It was shown that mesodermal competence is
lost not only in isolated ectoderm, but in dissoci-
ated division-arrested cells on the same time sche-
dule as in whole embryos [22]. Loss of neural
competence, and the gain and loss lens compe-
tence can all occur in isolated ectoderm. The
major conclusion is that these changes in compe-
tence take place as a result of intrinsic and auto-
nomical processes, occuring within the cells of
TABLE 3. Comparison of time (min) of cell cleavage ( 7) and somite formation (Ts) at
identical temperatures in embryos of three salmon species’ (Salmonidae)
Species ane Chum Rainbow’
Temperature To Ts T Ts To Ts
(Ke
3.0 657 670 570 654 514 S27)
4.0 558 555 480 538 433 431
5.0 479 470 408 452 367 357
6.0 415 397 350 374 314 300
7.0 364 328 303 317 270 254
8.0 321 284 265 270 234 219
9.0 234 232
+° 16.7 3.9 V3) 16.9 20.9 4.6
' The value of to and rs are obtained by nonlinear regression [35] from the own data.
The value of to for rainbow trout embryos is calculated from the data of Ignatieva [36].
1108
MI
Y. N. GorRoDILov
|
100
34 42 50 hr
584
592 600
Fic. 3.
608 616 624 hr
Comparison of the duration of cell cycles of the first five cleavage divisions and of the formation of five
successive somites (from 19th to 23rd) during linear somitogenesis in Atlantic salmon embryos at constant
temperature. Horizontal axis (a, b), time of incubation at 4.8°C from the moment of fertilization. Vertical axis,
(a) changes of mitotic index (MI) (data of Gorodilov and Lilp, 1978).
TABLE 4. Time of somite formation (zs) in pink and masu embryos (Pacific salmons from
genera Oncorhynchus) and their hybrids at identical incubation temperature
Value of ts (min)
Vener Temperature
b ti of incubation 2 pink 2 masu
obsenvallon (C, +0.05) pink masu x x
masu pink &
1987 125) 148 137 — 149
1988 6.6 327 310 314 —
1989 4.6 481 — 458 —
1989 6.4 344 — 333 —
1989 12.4 150 = 140 —
1990 5.9 379 = 368 _
ectoderm [23]. Besides, in some experiments it
was observed that inducing and reacting abilities of
developing tissues and cells gain or loss abruptly
[23, 44-45]. As Gurdon [41] has emphasized, in
the analysis of induction it is important to be able
to analyse a rapid response that can be observed in
single cells. It may be presumed that the changes
in development occur in discrete steps the duration
of which is equal or proportional to zo and ts.
THE ROLE OF DEVELOPMENTAL CLOCK IN
THE REGULATION OF EMBRYOGENESIS
The evidence of rhythmic processes occuring at
various stages of embryogenesis definitely indi-
cates the presence of a time control mechanism in
the embryos. The time count probably begins at
the moment of fertilization and might continue
Rhythmic Processes in Embryogenesis 1109
TABLES. Determination of earlier cleavages duration and to values in embryos of pink, masu and
their hybrids at temperature 10.1°C
Time. (min)’ from insemination up to Duration of one cell
Species Numbecot beginning in 50% embryos cytokinesis of cycle to (min)
et the cubis first second forth Fe ee
Sony Os aes cleavage cleavage cleavage Tu T1 aa
T] TIv
pink 113 Se3)3E 2.3) 765 +2.8 1240+3.5 232+1.8 236+1.2
masu 125 SiS)ae2,/ 780+1.9 1199+3.6 205+1.6 208+1.3
2 pink
Xx 109 585+4.9 802 +3.8 1245 +2.7 217+1.7 220+1.4
masu
9 masu
x 116 573 +4.0 790 +3.5 1250+3.3 NG Pew 226+1.6
pink ff
" The deviations of the data was estimated by the Berehns formula [40]
until the end of embryogenesis. We consider this
mechanism to be responsible for the general sche-
dule of development. It keeps all processes within
particular time limits. This system can be imagined
as a kind of metronom with oscillation period
equal or proportional with ration 1:1, 1:2, 1:3,
etc. to the duration of different processes, steps
and stages of differentiation and morphogenesis,
oscillations of which influence all embryonic cells.
We suggest that in the egg the signals which are
received by all embryonic cells are generated
periodically and serve to switch on/off expression
of genes (or clusters of genes), programmes and
subprogrammes of differentiation and mor-
phogenesis in various types of cells and parts of the
embryo depending on the serial number of a
particular signal. This endogenic pacemaker with
regular time intervals triggers the development of
cells and multicellular structures in different pre-
set directions.
We tried to show this scheme of development in
Fig.4. The time schedule of the development
beginning from fertilization with (zo or Ts if these
intervals are equal) as a time unit is shown on the
left. The stages of development of some
embryonic structures, parts, organs which are
started or finished simultaneously, i.e. at one and
the same controlling signal of a clock are indicated
at a horizontal line. This scheme is based on our
data on embryo development in salmon fishes. For
example [3, 33] during the first zt intervals the
cleavage divisions occur, after 12 rz from the start
of development blastula with enveloping and deep
cell layers and periblast is formed, after 56 r the
axial organs are formed and somitogenesis begins,
etc. By 350 +t embryogenesis is completed in
general and morphologic development is mostly
over.
According to this scheme the developmental
programmes for various differentiations are re-
leased in time by the embryo clock so that each of
them is switched on according to the serial number
of a clock signal. Under such condition, various
programmes can be completed independently.
This would explain the fact that some cyclic pro-
cesses in embryonic cells continue when cyto-
kinesis or nuclear divisions are suppresed [26].
This can be also the cause when isolated cells and
tissues of cartilage [27] or of amphibian ectoderm
[22, 23] kept their developmental schedule by the
same way as if it had been happened in the intact
embryos. At the same time some processes are, of
course, interdependent due to induction, hormo-
nal and other interactions in the embryo.
What happens if the formation of a structure is
not completed by the time of a new signal? Prob-
ably, in the case of vitally importnat organs (heart,
brain, kidneys) it is lethal to the organism, but in
the case of less important organs the resulting
abnormality can be retained and at the new time
signal the organ enters the next stage of develop-
ment. For example, when deformations were
produced in somites by heat shock, further de-
velopment of somites continued normally and with
1110
i)
MEIN SS
PININASS :
Y. N. GoropILov
Number
of cells
TAINS :
enveloping deep cell 2000
D cell layer layers periblast
peri- germ ring outgerm germ 25000
derm cells cells cells
33
Ist
ecto- neural noto- meso- somite ento-
56 derm cord chord derm pair derm
18th prone-
brain spinal otic somite heart gut phros
3 cord cord vesicles pair anlage anlage anlages
30th differentiation
fore- hind- heart gill somite of gut of pro-
85 brain brein tube anlages pair nephros
Fic. 4. The scheme of salmon embryos development using z as a time unit (see explanation in the text).
normal rate [46, 47].
Thus, the time factor in animal embryogenesis is
not only one of the coordinates of the process of
organism formation, but it is a primary factor
actively monitoring the development with the help
of periodically generated endogenic rhythms. The
latte provides the mechanism which measures the
embryo inner time and thus defines the order of
functioning of embryo cells and multicellular struc-
tures.
In contrast with the formed adult organisms in
which timing is based on periodic astronomic pro-
cesses and hence is common in all species, we think
the time in embryos is counted by a species-specific
temperature-dependent internal periodic process
(of unknown nature). After completion of
embryogenesis the individuals gradually acquire
outer universal rhythms which direct their living.
ACKNOWLEDGMENTS
I would like to thank Ms. Tamara N. Svimonishvili for
the help in the experimental work, Prof. G. V. Lopashov
and Prof. L. N. Seravin for useful theoretic discussions
and critically reading the manuscript.
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ZOOLOGICAL SCIENCE 9: 1113-1126 (1992) © 1992 Zoological Society of Japan
REVIEW
What is a Classification?
A Case Study in Insect Systematics: Potential
Confusion before Order
Kunio Suzuki! and Davip G. FurtH?
"Department of Biology, College of Liberal Arts, Toyama University, Gofuku,
Toyama City, 930 Japan; Museum of Comparative Zoology, Harvard
University Cambridge, Massachusetts 02138, U.S.A.
ABSTRACT—A case study is presented of the Leaf Beetle genus Orthaltica (Coleoptera: Chrysomeli-
dae) as an example of a group involved in a variety of taxonomic problems and controversies
demonstrating many of the technical and philosophical complexities of systematics that often make the
derivation of a classification difficult and confusing. The history of Orthaltica is reviewed providing
discussion and illustration of the concepts of: multiple synonymy and homonymy; the genus and
subgenus; lumping and splitting; and a systematic character, especially morphological, and its
application in formulating higher classifications. Based on morphological characters Orthaltica
provides a good vehicle for considering the classification of the two largest subfamilies of the Leaf
Beetles, the Galerucinae and the Alticinae (approximately 500 genera each). The primary difference
between the Galerucinae and the Alticinae is the absence or presence, respectively, of the jumping
organ (metafemoral spring), although some secondary differences exist in varying degrees. These two
chrysomelid subfamilies are so diverse and relatively unstudied systematically that deriving a higher
classification or speculating on their phylogeny, including their derivation from one (monophyletic) or
many (polyphyletic) lineages, is premature and needs a considerable amount of study of many
characters.
INTRODUCTION
One purpose of systematics is to recognize the
existence of all organisms on the earth, to describe
and order them, and to establish a good classifica-
tion system. A well-organized classification system
may be useful in deepening our understanding of
the organic world. If we have no reliable system
established by systematists, we cannot find any
order in the biologically diverse organic world.
This can be easily understood if we go to a zoo, a
botanical garden or a natural history museum. If
animals and plants are displayed without any con-
sideration of their inter-relationships, we know
only that these various organisms exist. The
inter-relationship among organisms is the key to
Received August 19, 1992
organizing them. What is the “inter-relationship”
among organisms? The authors use this term in
the sense of evolutionary relationship; that is, they
assume that all organisms on the earth have evolu-
tionary relationships, and that they are descended
from the first organism about 4 billion years ago.
One purpose of systematics is to organize all
organisms by such evolutionary relationships, and
even though exact evolutionary history may be
impossible to reconstruct completely, we can use
systematic methods to make limited approxima-
tions. The authors do not adopt the viewpoint of
numerical taxonomy (phenetics) which generally
ignores historical perspectives. The authors also
do not completely follow cladistics, a type of
systematics which often forces taxonomists to
make premature choices based on a restricted
methodology oriented toward producing phy-
1114
logenetic (lineage) results. In this context the
authors follow more the methodology of ‘evolu-
tionary systematics’ (sensu Mayr, 1963 [1]). Evo-
lutionary relationships may be recognized based
on close analysis of systematic characters in a
broad sense. Much of this paper will address the
question “what is a systematic character?” and its
application.
What is a phylogenetic classification? Most of
the problems discussed here are interconnected
and relate to this question. To answer this ques-
tion is not a simple matter, as with a dictionary
difinition, but rather a complex multi-faceted issue
with arguably different viewpoints concerning both
present and past relationships of organisms. We
attempt here to recognize the various aspects and
problems in answering such a question. The term
“phylogenetic classification” in this question refers
to a classification of a particular group which will
reflect evolutionary history. Readers should note
that the authors use the term “phylogenetic” in-
stead of “natural” classification. This is a con-
scious usage in order to avoid confusion because
the term “natural classification” has also been used
by numerical taxonomists in a restricted sense to
mean a clustering of groups based on overall
similarities of many characters. The authors’ usage
of this term (natural classification) actually agrees
more with its usage by cladists. One goal of
systematics, and the taxonomists who practice it, is
to establish a better phylogenetic classification
system which will be acceptable to more system-
atists and will result in a more consistant under-
standing and usage of scientific names for taxa.
One of the intentions of this paper is to demons-
trate to other non-systematist biologists and other
scientists some of the issues, problems and logic
present in the field of taxonomy/systematics.
Although the examples used from insect (beetle =
Coleoptera) systematics are at times somewhat
complicated and some problems are left unre-
solved, nevertheless, they illustrate some of the
fundamental complexities of biological system-
atics. In this paper the authors discuss some of
their opinions about phylogenetic systematic proc-
edure (theory and methods) based on recent stu-
dies. As with most basic systematic research, the
authors are studying a specific group, the Leaf
K. SUZUKI AND D. G. FurtH
Beetles (Chrysomelidae) from a particular view-
point. In systematic studies there are different
problematic situations with theoretical and/or
methodological viewpoints that differ from syste-
matist to systematist and from taxon to taxon. This
is occasionally an important obstacle to com-
munication among systematists. Using these spe-
cific studies, the authors attempt here to present
their awareness of the issues in systematics. The
authors would also like to help correct or improve
the present unfortunate situation which underesti-
mates the role and importance of systematic study
as compared with other experimental biological
disciplines. Such an attitude is based on scientific
ignorance and/or a glaring misunderstanding or
prejudice about the practice and significance of
systematics. Unfortunately there are even some
systematists who do not understand the signi-
ficance of systematics. This is all the more reason
for a clear and consistent explanation of biological
systematics and its role in basic science. In order
to stimulate further development of biosystema-
tics, it is essential to cooperate with other biologic-
al disciplines. This has been occasionally pointed
out but has not been practiced effectively. One
possible reason for this scarcity of interdisciplinary
cooperation may be the lack of mutual awareness
of the issues existing between systematics and
those other disciplines. Systematists are at least
partially to blame for this because they have not
often stated their awareness of such interdisciplin-
ary issues or the significance of systematics as a
basic empirical science, and as a stepping stone to
other modern aspects of biological sciences.
The authors have recently conducted research
together concerning several topics in insect sys-
tematics. There are even differences in theoretical
and/or methodological approaches between the
authors even though they both have studied the
same insect group for many years. However, it has
become increasingly apparent through discussion
and mutual criticism that both authors have a
common recognition of the above-mentioned
problems of cooperative efforts in systematics.
Through this mutual and reciprocal interaction the
authors have managed to complete many systema-
tic studies within a relatively brief period that
could not have been accomplished in the same
What Is a Classification?
broad-spectrumed way, if each of us had studied
the same topics individually. The authors hope
that such cooperative studies will serve as an
example to systematists studying different groups
or using different methodologies, and to biologists
of different disciplines who might mutually augu-
ment their individual research through cooperative
discussion and studies.
In this paper, we are going to focus on a problem
which we call the “GA Problem”. G and A refer
to the chrysomelid subfamilies Galerucinae and
Alticinae, respectively. The core of the “GA
Problem” may be summarized as follows: “How
can we classify the Galerucinae and Alticinae?” or
more specifically “How should the Galerucinae
and Alticinae be defined?” —this form of question
may show that this problem is one of group/taxon
recognition or establishment. Most systematists
have accepted the view that the family Chrysomeli-
dae should be classified into 19 subfamilies; Gale-
rucinae and Alticinae are the largest. Various
opinions have been proposed concerning the sys-
tematic position or phylogenetic relationships of
these two groups (e.g. Crowson, 1955, 1982 [2, 3];
Lawrence & Britton, 1991 [4]) but it is generally
accepted that they are closely related to each
other. However, within these two groups there are
several taxa (mostly genera) whose true systematic
position (even subfamily placement) has not been
decided. So that, the above question may be
better expressed as: “How should the Galerucinae
and Alticinae be distinguished?”. There is a
variety of possible ways to express this problem
depending on differences in the awareness of the
issues and/or which points are emphasized. Var-
ious practical aspects derived from this GA Prob-
lem may be included in this type of question. The
authors will introduce these aspects using exam-
ples wherever possible because they involve many
typical problems encountered in systematic study
of other organisms.
Systematics has, of course, an essential purpose
to deepen our recognition of a particular toxon.
However, if we are satisfied with only this aspect of
systematics one might ask “why is systematics
needed?”, especially when such a huge number of
organisms exists on the earth. In other words, if
we say that a purpose of systematics is to know all
1115
organisms, is this not an endless task, how can this
be sensibly planned and how can this survey of
organisms be justified? Such questions are often
used as arguments against systematists; Sibatani
(1960) [5] asked such an essential question to
systematists in Japan. Some systematists may
reply that the people who laugh at such a biotic
inventory do not know the world and they cannot
understand the significance of organic diversity on
our earth. Yet systematists are convinced that
giving meaning to organic diversity on the earth
from various aspects should be one of the most
important priorities in biology.
Even though systematics has its own issues and
perspectives, it also gives a kind of a bird’s eye
view to other biological disciplines. The bird’s eye
view which systematists offer resembles a topo-
graphical map. The precision (scale) of the map
depends on the area (organic group). If we do not
have any map, we cannot walk even one step. At
the same time, even if we have a map we may lose
our way because of disagreement between the map
and our present spot. We must make an effort to
improve the map in order to reflect our present
position; this analogy resembles the relationship
between systematics and other biological disci-
plines. The opinion that systematics is a synthetic
discipline in biology may have originated from the
viewpoint that both systematics and other biologi-
cal disciplines should be mutualistic.
survey of our present position will be necessary in
order to improve the map to the point of maximum
usefulness. The opinion that we do not need so
many kinds of organisms for biological research
shows gross misunderstanding of science. Biolog-
ical inventory studies have limitations and, be-
cause of rapid destruction of natural ecosystems by
humans in recent years and the resulting extinction
of many organisms, there is an intense crisis and
urgency that systematists realize more than any
scientists. Systematists are the only ones who can
record the great biological diversity that exists and
that which is being lost forever. Therefore, system-
atists should play a leading role in alerting others
(scientists, politicians, etc.) to the current crists
and in devising plans for conservation of this
organic diversity.
Extensive
1116
GA PROBLEM
Orthaltica: History and Confusion
In order to consider “GA Problem” (Galeruci-
nae— Alticinae Problem) the authors have chosen
the genus Orthaltica as a good example to demon-
strate many of the problems of systematics, begin-
ning with a brief historical review. The authors
would like to give an explanation of the present
problem based on Scherer (1974) [6] and using the
following table (list of synonymous names) from
his article:
Genus Orthaltica Crotch, 1873
Orthaltica Crotch, 1873:69 (type-species:
Crioceris copalina Fabricius; N. America);
Horn, 1889 :236, 247; Blatchley, 1910: 1206,
1215; Heikertinger, 1924-25 (1925): 65;
Arnett, 1963 :914, 938.
Leptotrix Horn, 1889 : 236, 249 (type-species: L.
recticollis LeConte; N. America (nec Menge,
1868; Araneae) see Leptotrichaltica.
Livolia Jacoby, 1903:15 (type-species: L. sulci-
collis Jac.; Africa); Scherer, 1961 :268: 1969:
10, 19, 118, 242; 1971: 1-37. New Synonym.
Leptotrichaltica Heikertinger, 1924-25 (1925):
68 for Leptotrix Horn. New Synonym.
Micrepitrix Laboissiére, 1933 : 205 (type-species:
M. coomani Lab.; Tonkin); Gressitt, 1955 :35
(Alticinae); Gressitt & Kimoto, 1963: 404,
575; Samuelson, 1965 :215; Scherer, 1969 : 10,
19, 98; 1971: 10 (as synon.)
Serraticollis B. E. White, 1942 : 17 (type-species:
S. rhois White; Calif.); Arnett, 1963 :938 (as
synon. )
The essence of the history of systematic treat-
ment of Orthaltica is compiled in the above table.
A trained systematist can easily understand not
only the formal meaning but also the fact that
beyond the nomenclature this group may contain
many systematically difficult problems. The main
information that should be deduced from this table
may be arranged as follows (supplemental com-
ments in brackets).
This genus was established by Crotch (1873) [7]
based on Crioceris copalina described by Fabricius
K. SUZUKI AND D. G. FurtH
from North America. [The genus Crioceris cur-
rently belongs to the subfamily Criocerinae.
Crotch considered O. copalina as a member of a
previously unknown genus currently in the Altici-
nae. However, even by Crotch’s time higher
classification of the family Chrysomelidae had
been only gradually and not well established.
Chapuis (1874) [8] was the first to propose a higher
classification system; this was the basis of our
modern system. However, not until Jacoby (1908)
[9] was an actual subfamily system established. So
that, we should understand that Crotch regarded
the species copalina as a member of a close relative
of many genera which are included in the Alticinae
today]. After Crotch, Horn (1889) [10], Blatchley
(1910) [11], Heikertinger (1925) [12], and Arnett
(1963) [13] followed his treatment. [This also
means that all of them recognized the genus
Orthaltica as valid].
Until now the following have been considered
synonyms of Orthaltica:
Leptotrix: This genus was established by Horn
(1889) [10] based on the species recticollis de-
scribed by LeConte from North America. Howey-
er, this genus name (Leptotrix) was a homonym, in
other words, the name was already preoccupied by
Menge (1868) [14] as a name of a spider genus (see
also Leptotrichaltica). [Here, some readers may
think that Horn should have recognized the inde-
pendence of Orthaltica from Leptotrix within his
1889 article [10]. This suggests the following two
possibilities: (1) Horn positively recognized the
independence of both genera or (2) he established
Leptotrix because he did not recognize the identity
of Orthaltica. In order to judge which possibility is
probable, we have to examine Horn’s 1889 paper
[10]. In this paper, Horn pointed out that Orthalti-
ca was similar to other genera like Crepidodera
and Pseudoepitrix. He then described Orthaltica
melina as a new species and, following the descrip-
tion of O. melina, he established Leptotrix and
pointed out that this new genus resembles Orthalti-
ca and Pseudoepitrix but did not belong to any
genus of the tribe Crepidoderides. Subdivision of
Alticinae into tribes has been partially attempted
by Leng (1920) [15] and Bechyné and Bechyné
(1975) [16] but remains very confusing and invalid.
According to the above facts, the authors judge
What Is a Classification?
that Horn took the first possibility mentioned
above. Moreover, we should pay attention to the
fact that in Horn’s era a genus was likely to have
been defined more typologically than in recent
times. In other words, a genus was established
based on a comparison with type-species and, in
general, it was likely to be more subdivided. Of
course, this may vary from worker to worker and
from group to group].
Livolia: This genus was established by Jacoby
(1903) [17] based on a new species sulcicollis from
Africa. Scherer (1961, 1969, 1971) [18, 19, 20]
followed this but synonymized it with Orthaltica in
his 1974 article [6].
Leptotrichaltica: Heikertinger (1925) [12] gave
a new name to Leptotrix Horn because the exis-
tence of a homonym (mentioned above); this
genus was also synonymized with Orthaltica by
Scherer (1974) [6].
Micrepitrix: This genus was established by
Laboissiére (1933) [21] based on the type-species
M. coomani from Tonkin. [Laboissiére described
this genus as a member of the Galerucinae but
Gressitt (1955) [22] transferred it to the Alticinae.
After that, Gressitt & Kimoto (1963) [23], Samuel-
son (1965) [24], and Scherer (1969) [19] followed
this, but Scherer synonymized it with Orthaltica in
his 1974 paper [6]].
Serraticollis: This genus was established by
White (1942) [25] who described the type species
(S. rhois) from California. After that, Arnett
(1963) [13] synonymized this genus with Orthalti-
ca. |Though his opinion cannot be determined
from this synonym list, this was suggested to
Arnett by J. A. Wilcox, an authority of the Gale-
rucinae]. The items mentioned above are the
things which are summarized in Scherer’s (1974)
[6] synonym list. One should examine further each
of the previous worker’s opinions; this is an impor-
tant routine for many systematists.
The authors will now examine some of the
details of the confusion in the systematic treatment
of Orthaltica. Scherer (1974) [6] synonymized five
genera with Orthaltica. In considering whether his
treatments are reasonable or not one has to ex-
amine all the literature involved and often to
examine original type-specimens, depending on
the situation. Seeno and Wilcox (1982) [26] listed
1117
Leptothrix Heikertinger et Csiki (1940) [27] as a
synonym of Orthaltica; however, such a differece
in spelling between Leptotrix and Leptothrix is not
significant. Because Leptotrix is a homonym of a
spider genus, the species belonging to it should be
automatically transferred to Leptotrichaltica estab-
lished by Heikertinger (1925) [12] as a new name
for Leptotrix. This is merely follows proper sys-
tematic treatment in accordance with the interna-
tional rules of zoological nomenclature. We have
to consider the other four genera Livolia, Micr-
epitrix, Leptotrichaltica and Serraticollis, the first
three of which were synonymized with Orthaltica
by Scherer (1974) [6].
1. Livolia:
When this genus was established, Jacoby (1902)
[28] mentioned that this genus may be transitional
between the Alticinae and Galerucinae. In his
revisional study of this genus, Scherer (1971) [20]
treated the following 20 species, which included
seven known and 13 new species. Also in this 1971
paper Scherer synonymized Micrepitrix with Livo-
lia species ([20]; see also Scherer, 1981 [29]). In
the list below “nov. comb.” (new combination)
means a new change of genus assignmnet of the
species in question, “nov. spec.” (new species) a
new species is described and in brackets is the
original locality (type-locality).
vestita (Baly, 1877) nov. comb. [W. Australia]
sulcicollis Jacoby, 1903 [Mashonaland: Salis-
bury]
“africana nov. spec. [W. Africa]
coomani (Laboissiére, 1933) nov. comb. [China:
Tonkin; Hainan I.]
carolina (Chuaj6, 1943) nov. comb. [Yap; Palau]
minuta (Jacoby, 1887) [Ceylon]
*minor nov. spec. [Singapore]
*fulva nov. spec. [W. Sarawak]
*nigripennis nov. spec. [Singapore]
*sarawakensis nov. spec. [W. Sarawak]
“parva nov. spec. [Singapore]
*malayaensis nov. spec. [Malaya]
*perakensis nov. spec. [Malaya]
*assamensis nov. spec. [India: Assam]
*serraticollis nov. spec. [Burma] [=impres-
siceps: Scherer, 1974]
*tenasserimensis nov. spec. [S. Burma]
1118
*ceylonensis nov. spec. [Ceylon]
minutiuscula (Csiki, 1940) [Sumatra]
*Jaticollis nov. spec. [Singapore]
laboissierei (Chen, 1935) nov. comb. [E. China:
Kiangsi]
Of these 20 species, we exclude here the 13
species with asterisks described by Scherer in this
paper [20] from our present discussion because
they were originally described as members of
Livolia; we will examine the seven remaining
species. Naturally in such cases systematists have
to examine all the original literature involved,
even though in a practical sense this is sometimes
quite difficult for taxonomists who do not have
easy access to good libraries.
vestita: This species was described by Baly
(1877) [30] as a member of Crepidodera. This
means that in Scherer’s opinion at least some
species which belong to Livolia have been mixed in
Crepidodera (see also Scherer, 1982 [29]).
sulcicollis: This is the type-species of Livolia.
Thus, as far as the genus continues to exist this
species plays an important representative role.
coomani: This species was described by Laboi-
ssiére (1933) [21] as a member of Micrepitrix. If we
regard this species as a member of Livolia, this
means that at least some species of Livolia have
been mixed in Micrepitrix. If we agree with
Scherer’s (1971) [20] opinion that Micrepitrix is a
synonym of Livolia, this problem is eliminated
immediately; this is discussed below.
carolina: This species was described by Chuj6
(1943) [31] as a member of Epithrix (= Epitrix). If
we regard this species as a member of Livolia, this
means that some species have been mixed in
Epitrix. Gressitt (1955) [22] treated this species as
a member of Micrepitrix. Therefore, as in the case
of coomani, at least some species which should
belong to Livolia have been mixed in Micrepitrix.
Here, we experience the following two derived
problems: (1) what kind of systematic relationship
is there between Epitrix and Livolia and (2) how
should we treat the systematic position of carolina.
For the first problem, one possible answer may be
that if Micrepitrix should be regarded as a synonym
of Livolia, we have to consider systematic treat-
ment of Epitrix separately; that is, because Epitrix
K. SuzuKI AND D. G. FurtH
is a very big group, even if there are systematic
changes for some species treated as members of
this genus, all other species are not necessarily
transferred to Livolia. For the second problem, as
with other species which have been treated as
members of Micrepitrix, the true systematic posi-
tion of this species cannot be determined automati-
cally.
minuta: This species was described by Jacoby
(1887) [32] as a member of Crepidodera and, thus,
the same situation as in vestita can be pointed out.
Scherer (1969) [19] already pointed out that this
species should belong to Livolia.
minutiuscula: Concerning systematic treatment
of this species, there is a problematic history. The
author of this species is Csiki (1940) [33] in Heiker-
tinger and Csiki (1940) [27]. But, the taxon
corresponding to this species was first described by
Jacoby (1895) [34] under the name of minuta as a
member of Crepidodera. But, as above, Jacoby
(1887) [32] already used the name minuta as a
member of Crepidodera. That is, Jacoby produced
a new homonym to his other species in the same
genus! This is a very unusual example of a
homonym. By discovering this fact, Csiki (1940)
[33] became the author of this species without
describing even one line about this species.
laboissierei: This species was described by Chen
(1935) [35] as a member of Micrepitrix. This is the
same situation as in the above cases of coomani
and carolina.
Scherer (1974) [6] changed the name of his L.
serraticollis to impressiceps because of homonymy;
that is, the name was preoccupied by Samuelson
(1965) [24] as a name for one of three species of
Micrepitrix from New Guinea. Such homonymy
produces a quite confusing history that seems to be
mysterious for workers of other fields of biology.
The authors summarize above items as follows.
In analyzing Scherer’s (1971) [21] opinion, the
systematic relationships between three genera
(Crepidodera, Epitrix and Micrepitrix) and Livolia
are: (1) in the first two genera, only some species
belonging to Livolia had been mixed; so that, their
systematic assignment to a genus needed to be
changed and; (2) Micrepitrix should be regarded as
a synonym of Livolia; so that, all species described
as members of Micrepitrix should be automatically
What Is a Classification?
transferred to Livolia. After all these changes,
Scherer (1974, 1982b) [6, 29] ultimately regarded
Livolia as a synonym of Orthaltica.
2. Leptotrichaltica:
Concerning the systematic treatment of this
genus, we completely agree with Scherer’s opinion
that it should be regarded as a synonym of Orthal-
tica. That is, Leptotrix (=Leptotrichaltica) recti-
collis described by Horn (1889) [10] can be treated
as a member of Orthaltica according to the original
description.
3. Micrepitrix:
Sherer (1971) [20] synonymized this genus with
Livolia. Samuelson (1973) [36] followed his opin-
ion. Because of several reasons mentioned below,
the authors would like to treat Livolia and Micre-
pitrix as independent genera, not as synonyms.
4. Serraticollis :
This genus was treated by Arnett (1963) [13]
(following Wilcox’s opinin) as a synonym of
Orthaltica. The authors agree that White’s (1942)
[25] original description of this genus can be com-
pletely adopted into Orthaltica.
Genus vs. Subgenus/Lumping vs. Splitting.
Scherer (1971, 1974, 1982, 1988) [20, 6, 29, 37]
synonymized Livolia and Micrepitrix with Orthalti-
ca. The first two genera have a symmetrical
aedeagus, whereas Orthaltica as an asymmetrical
one, which is extremely rare in the Alticinae. Of
course, Scherer recognized this fact and main-
tained that such differences should be regarded as
the characters of a subgenus level. The basis of his
assertion seems to be his special concept of a
genus. In his 1973 paper [38], Scherer pointed out
“how reliance on topological criteria and over-
ranking of lesser units can destroy the phylogenetic
image of a genus.” He also regretted that many
systematists are likely to treat closely related spe-
cies groups as independent genera based only on
morphological characters. According to Scherer,
Livolia and Micrepitrix should not be separated
from one another (the latter, which was estab-
lished more recently than the former, is regarded
as a junior synonym) and neither should be distin-
guished from Orthaltica at genus level. Thus, this
Livolia-Micrepitrix complex is regarded as a
synonym of Orthaltica and should be distinguished
1119
from Orthaltica in the strict sense (Orthaltica (s.
str.)) only at the subgeneric level as Orthaltica
(Livolia). In the case of Orthaltica, if one makes a
classification based on Scherer’s broadened con-
cept, he would be considered a “lumper” (i.e.
joining groups together); in contrast to this, many
systematists are “splitters” (i.e., dividing groups
apart). Occasionally the distinction between the
so-called “lumper” and “splitter” has been discus-
sed in systematics; it is, of course, a somewhat
relative and subjective matter.
The authors would like to point out the follow-
ing two problems. First, Scherer (1973, 1982,
1988) [38, 29, 37] suggests that taxonomists use a
broadened interpretation of the genus by “lump-
ing” subunits which share common ecology and/or
historic-zoogeography into subgenera rather than
“splitting” them into several genera. Although
Scherer agrees that the genus should include spe-
cies of common ancestry (monophyletic), he con-
siders that interpretation of the “gap” separating
higher categories is a critical aspect of defining
generic level ranks (i.e. genera or subgenera).
However, even as Scherer partially admits, the
information (e.g. ecology, historic-zoogeography,
etc.) mecessary to define a genus is not always
available for many groups; on the contrary, it is
often quite limited. Although the authors agree in
principle with Scherer’s (1973) [38] definition of
the genus concept, we suggest that some of his
examples of genera (especially Orthaltica) are too
broadly defined (“lumped”) and may be as difficult
to interpret evolutionarily as with genera which he
claims are over-split.
Secondly, several items that may lead to diffe-
rent systematic treatment should be considered.
Synonymyzing of genus A with genus B will result
in automatic transfer of the species members of A
to B. This also means automatic extension of
category A. In the present case, if Livolia and
Micrepitrix are synonymized with Orthaltica, this
automatically extends Orthaltica into having a very
wide geographical range. On the contrary, if we
regard these three as independent genera, we
understand their distribution as follows: Orthaltica
from North America, Livolia from Africa, and
Micrepitrix from Southwest Asia to Pacific. Of
course, comprehension of such facts are important
1120
for us and although some may consider that such a
divided treatment of ranking in a taxon is essential-
ly not important, this demonstrates a gross mis-
understanding. For example, when we consider
phylogenetic relationships at the genus level, from
the lumper’s viewpoint (Scherer, 1974, 1988) [6,
37] the differences among these three groups
(Orthaltica, Livolia, Micrepitrix) may be regarded
as intra-generic variation, whereas for the splitters
they may be viewed at the inter-generic level. This
may be deduced by an ordinary comparative
method. Suzuki (1984, 1989a) [39, 40] has pointed
out that the ranking of any taxon must strongly
influence the phylogenetic consideration of that
group.
Lumping and splitting have both good and bad
points. Actually, one should judge individual
cases of taxon ranking according to the differences
in the effect on the understanding of the groups in
question. Our conclusion about this problem is
that in a group for which there is a wealth of
information, Scherer’s (1973) [38] broadened
treatment of the genus (using subgenera) may help
the evolutionary understanding; however, in cases
where there is not much information about the
group in question, Scherer’s lumping treatment
can have a negative effect on the evolutionary
understanding. Unlike some systematists the au-
thors do not think that splitting is an improper
systematic practice. Rather we recognize the na-
ture of each of the groups which we study and
prefer to enhance the knowledge of each group by
finding and analyzing differences between/among
them. Essential characteristics and relationships of
a given group can be obscured by the lumping
treatment.
Concerning the Livolia-Micrepitrix-Orthaltica
problem discussed in this paper, unfortunately
there is currently only limited information about
them. A lumping treatment of these genera into
Orthaltica would indicate their general similarity
but the individual characteristics and differences of
each of three groups would be hidden. Therefore,
at this relatively initial stage, based on differences
in the aedeagus and geography and in the interest
of clarify of these entities for future evolutionary
analysis, the authors prefer to treat Livolia, Micr-
epitix, and Orthaltica as three independent genera.
K. Suzuxi AND D. G. FurtH
In the phylogenetic sense of cladistics, it is impor-
tant to establish that each genus has evolved from
a single ancestral lineage or clade, i.e. monophyle-
tic. The above examination of the problematic and
complex situation of the genus Orthaltica has gra-
dually unfolded to the authors and based on it we
now begin to approach the core of GA Problem.
Concerning the higher systematic position of
Orthaltica, there is a difference of opinion among
some chrysomelid systematists, i.e. even its sub-
family placement (Furth, 1985, 1988 [41, 42];
Reid, 1990, 1992 [43, 44]).
Recognition of Higher Taxa in Chrysomeloidea.
Mainly based on extensive comparative morpho-
logical study of the internal reproductive systems
of both sexes, Suzuki (1988) [45] pointed out that
the family Chrysomelidae cannot be regarded as a
monophyletic group in relation to the two other
familes Cerambycidae and Bruchidae of the super-
family Chrysomeloidea. Suzuki's first essential
question about chrysomeloid phylogeny concerned
the fact that, surprisingly, the Chrysomelidae
(Leaf Beetles) cannot always be clearly distin-
guished from the Cerambycidae (Long-horned
Beetles) and Bruchidae (Seed Beetles); he natural-
ly assumed that there must be greater differences
among higher categories than among lower ones.
The classification at subfamilial level in these three
families has been considerably well established and
consistent, although there are still differing opin-
ions. There are, however, relatively few obvious
differences that separate these three familes from
each other.
The assignment of a given species to any of the
three families is automatically determined by the
fact that the species belongs to a particular sub-
family within them; a kind of inverted funnel
system. Suzuki (1984, 1989a) [39, 40] pointed out
that this type of problem is one of essential weak
points in the Linnaean hierarchical classification
system and its concomitant hierarchic system of
category names. In most entomology textbooks,
one can find some characteristics to separate these
three families, and actually one can classify most
specimens/taxa into families, but there are several
exceptions. In a recent popular book, White
(1983) [46] mentioned that “(the family Chry-
What Is a Classification?
somelidae) cannot be readily characterized; family
members are very diverse and have no distinctive
characters in common.” This is a very basic
statement, but if true, how should we answer the
question “what is a Leaf Beetle or a Long-horned
Beetle or a Seed Beetle?” Actually beetle workers
have not really answered this question. For the
phylogenetic relationships in this superfamily, the
first author will give a general interpretation else-
where (Suzuki, in press [47]). The present GA
Problem is a smaller version of the same kind of
problems mentioned above; that is, GA Problem
involves the questions “what is Galerucinae?” or
“what is Alticinae?” or “how can one separate the
Galerucinae and Alticinae from each other?”.
There are probably similar situations in many
groups; however, in this case our current system of
recognition for these two subfamily groups is rel-
atively advanced.
Relationship of the Galerucinae and Alticinae,
and the MS Organ.
Howe have we separated the Galerucinae and
Alticinae so far? Actually there is a diagnostic
character, as indicated by the name “flea beetle,”
the members of the Alticinae can jump using a well
developed hind leg. They have a special jumping
organ, which has been known as Maulik’s organ or
metafemoral spring (MS) (Furth, 1982) [48] in
their hind femora (Fig. 1, [49]). In fact, the
scientific name of the type genus (Altica Fabricius)
is derived from the Greek ‘haltikos’ which means
good at jumping (Furth, 1988) [42]. Most chry-
somelid systematists have regarded the presence of
this MS organ as a diagnostic character by which
they can separate the Alticinae from the Galeruci-
nae. However, there are several potential excep-
tions to which this rule cannot be applied. The
genus Orthaltica represents just such an exception-
al group, because it lacks the MS organ in the hind
femora. The authors confirmed this fact in the
three North American species. If we follow the
tule strictly, the genus Orthaltica should belong to
the Galerucinae. Based on this fact Scherer (1974)
[6] and J. A. Wilcox (personal communication)
maintained that Orthaltica was an exceptional alti-
cine genus lacking MS organ. But Furth opposes
this point and has asserted that the genus belongs
1121
Fic. 1. The MS organ (Metafemoral Spring) of Nonar-
thra postfasciatum (Fairmaire, 1889) (Coleoptera,
Chrysomelidae, Alticinae). (a) Posterior view of
loft leg including the MS organ. (b) Enlarged MS
organ. Scale: 0.5mm. Taken from Suzuki and
Furth (1990b) [49].
to the Galerucinae (Furth, 1985, 1988, 1989) [41,
42, 50].
Before proceeding, the authors would like to
point out the following facts:
1. Besides Orthaltica, both Livolia and Micr-
epitrix also lack the MS organ (Furth & Suzuki, in
preparation).
2. The genus Eubaptus, the only member of
the subfamily Eubaptinae (Bruchidae), has the MS
organ (Teran, 1964, 1967) [51, 52]; the authors
have also confirmed this fact. The authors ex-
amined many other bruchids but could not find this
organ in any other groups (Suzuki & Furth, 1990b
[49]). In addition, the authors have examined
some Sagrinae species (Chrysomelidae), which
have been occasionally considered as close rel-
atives of the Bruchidae, and the genus Rhaebus
(Bruchidae, Rhaebinae), which has been treated
as a group of the Sagrinae, but has been transfer-
red to the Bruchidae (Crowson, 1946 [53]; King-
solver & Pfaffenberger, 1980 [54]; Borowiec, 1987
[55]); the authors could not find the MS organ in
either group.
3. There are species of the Rhynchaeninae
(superfamily Curculionoidea, family Curculioni-
dae) that have the MS organ. Maulik (1929) [56]
1122
mentioned that the metafemoral organ existed in
one species of the genus Rhynchaenus but that it
differed from that in the Alticinae. Pomorski
(1978) [57] first described this metafemoral organ
for another species of Rhynchaenus. Furth has
recognized a considerable intrasubfamilial (in-
tergeneric) morphological variation and disco-
vered seven different MS morpho-groups in the
Alticinae genera (Furth, 1980, 1982, 1985, 1988)
[58, 48, 41, 42]. The MS organ in the Rhynchaeni-
nae is quite similar to that in the Eubaptus and to
the most simple morpho-group in the Alticinae
(Furth & Suzuki, 1992 [59]).
The above facts indicate that the MS organ has
evolved independently in three phylogenetically
separated groups. Furth and Suzuki (1992) [59]
have discovered that it also exists in other coleo-
pterous groups. This suggests that these groups
may not have acquired this organ in their hind
femora in the same way, i.e., evolution of this
organ may have been regulated by the same de-
velopmental constraints but under different selec-
tive pressures. At the same time we must also
contemplate the reason why the MS organ did not
develop in other groups. In fact, there are various
coleopterous groups which have a developed
jumping ability in different ways (cf. Pomorski,
1983 [57], Furth & Suzuki, 1992 [59]). The MS
organ evolved from the sclerotization of the
metafemoral tibial extensor tendon (Furth & Suzu-
ki, 1990b) [61]. There are several non-jumping
beetle groups with well developed (swollen) hind
femora (Furth & Suzuki, 1990b) [61]; therefore,
the enlargement of hind femora is not always
concerned with a jumping function. The authors
have examined comparatively many insect orders
as well as beetles with enlarged hind femora from
the viewpoint of leg morphology (Furth & Suzuki,
1990a, 1990b) [60, 61].
The Galerucinae and Alticinae have long been
treated as independent subfamilies. Suzuki (1988)
[45] pointed out that there are few essential differ-
ences between them concerning much of their
internal reproductive systems; however, there are
some general differences between them in the
aedeagus (male) and the spermathecae (female).
For example, the aedeagus is usually asymmetrical
in Galerucinae and almost always symmetrical in
K. Suzuki AND D. G. FurtTH
Alticinae, the transverse spermathecal folds on the
spermathecal capsule are quite consistant in the
Galerucinae but variable in the Alticinae and the
bending of the basal part of the spermathecal
capsule (cf. Suzuki, 1988) [45] is distinctive in most
of the alticine genera.
Wilcox (1965) [62] mentioned that: “the Altici-
nae can be distinguished from Galerucinae by the
sclerotized extensor apodeme [=MS organ] in the
posterior femora of the former. Orthaltica and
Leptotrichaltica from the United States, Micrepi-
trix Laboissiére from the Oriental Region, and
Micrantipha Blackburn from Australia seem to be
the only exceptions to the rule. They appear to be
flea beetles which lack the extensor apodeme.”
Wilcox (1975) [63] also listed many genera, long
treated as the members of the Galerucinae, which
he claimed should be transferred to the Alticinae.
He considered the MS as the only character to
distinguish the Alticinae from the Galerucinae. It
should be mentioned here that recently Reid
(1990) [43] synomymized Micrantipha with Orthal-
tica as a subgenus of the latter. Reid included all
species of Livolia into Micrantipha (Micrantipha
was described in 1896 and, therefore, its name
takes precedence over Livolia described in 1903).
However, even more recently Reid (1992) [44]
suggests that his 1990 Micrantipha subgenus (in-
cluding the former Livolia and Micrepitrix) should
be restored to generic status. This is consistent
with the principles of the current paper as stated
above and previously (Suzuki & Furth, 1990a,
1990b) [64, 49].
Suzuki (1988, 1989b) [45, 65] emphasized char-
acter correlation among the phylogenetically im-
portant morphological charactesr. If we adopt the
MS organ as the only character to separate the
Galerucinae and Alticinae, at least the following
three assumptions should be valid:
1. The Galerucinae and Alticinae are each
monophyletic groups, we use this term in a cladis-
tic sense to show phylogenetic relationships.
2. The MS organ was acquired in the alticine
lineage after they diverged from the common
ancestor of the galerucine and alticine lineages.
3. In the alticine lineages species did not lose
the MS organ secondarily and similarly in the
galerucine lineages no species acquired the organ
What Is a Classification? 1123
secondarily. These assumptions mean that the MS
organ is a completely derived (apomorphic) char-
acter of the Alticinae. In other words, the mem-
bers of the Alticinae can be recognized by the
shared derived (synapomorphic) existence of the
MS organ. If these assumptions are valid, we
should be able to trace logically the transformation
process of the organ based on a comparison of the
existing species or groups. However, does the MS
organ guarantee us this ability? In other words,
does the MS organ reflect a clade (single lineage)
or a grade (group of lineages; cf. Huxley, 1957
[66])? If assumptions 2 and 3 are not valid, then
we cannot determine definitely whether Orthaltica
and the other two genera belong to the Alticinae,
an alticine lineage that secondarily lost its MS
organ, or the Galerucinae.
CONCLUSIONS
The diversity of Galerucinae (489 genera and
about 6,000 species—Seeno & Wilcox, 1982 [26],
Jolivet, 1987 [67]) and the Alticinae (more than
500 genera and approximately 8,000-10,000 spe-
cies (Seeno and Wilcox, 1982 [26]; Scherer, 1988
[37]) is so great that our current knowledge of most
aspects of relationships within each subfamily and
between them is very primitive. The variation is
extremely large in body size and form, color and
pattern, external and internal morphological char-
acters, as well as some important aspects of their
biology. Although the MS organ is currently a
useful taxonomic morphological character for
separating the Galerucinae and Alticinae as well as
for distinguishing and grouping Alticinae genera,
its evolution within the Chrysomelidae needs much
more study. Wilcox (1975) [63] listed 20 genera
formerly considered to be Galerucinae which he
transferred to other families or chrysomelid sub-
families. Most of these were transferred to the
Alticinae based solely on the presence of the MS
organ. This underlines the historical confusion of
these two obviously closely related chrysomelid
subfamilies.
The authors are beginning a long term study
using morphological character correlation, includ-
ing the MS organ, to elucidate the classification
within the Alticinae and their relationship to the
Galerucinae. Only after such a study is well
underway can we attempt to answer the questions
posed above about the assumptions for the phy-
logenetic relationship between the Galerucinae
and the Alticinae. Along with the questions raised
by assumptions 2 and 3 mentioned above, is the
question of assumption 1. In other words, can we
be certain that the Galerucinae and Alticinae are
monophyletic? If either or both subfamilies have
evolved from different (several) lineages (clades),
this would be considered paraphyletic (in a cladistc
sense) and would not be a valid unit for consider-
ing a phylogenetic analysis. However, there may
still be value in analyzing such a combination of
clades (cluster of groups or grades). This idea of
grades was discussed by Huxley (1957) [66] and
some systematists maintain that such grades also
reflect valid evolutionary processes (see Takagi,
1978) [68]. In fact, in many groups it is difficult to
clearly define clades or to be certain of monophly;
in other words, whether a group is composed of a
clade or several grades. This indicates one of the
problems with premature cladistic analysis.
However, as Huxley (1957) [66] pointed out, clade
and grade agree in many cases.
The GA Problem reflects a situation where,
based on our limited current state of knowledge, it
is not possible to make definite statements about
phylogeny or evolutionary relationships. Even
though there is theoretically no way at this point in
our knowledge of Galerucinae/Alticinae rela-
tionships to prove how the MS organ evolved or if
it could be secondarily lost, we must rely some-
what on what is known from an increasingly large
number of genera from both subfamilies. We must
proceed to study as many aspects (e.g. morpholo-
gy, ecology, genetics, etc.) of the relationship of
these two groups as possible with the ultimate goal
of confirming or negating the above three assump-
tions. However, the authors prefer to take a
conservative approach without over-speculation,
without lumping, and to consider the Galerucinae
and Alticinae as valid and separate subfamiles.
ACKNOWLEDGMENTS
We would like to thank the National Science Founda-
tion (INT9116359)and the Japan Society for the Promo-
1124
tion of Science for grants of the U.S.-Japan Cooperative
Science Program that enabled the authors to complete
this paper. The previous editor Dr C. Oguro (Toyama
University) kindly read our manuscripts and recom-
mended us to contribute to this journal.
10
11
12
13
14
15
16
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Proceedings of the
Sixty-Third Annual Meeting of the
Zoological Society of Japan
October 7-9, 1992
Sendai
1127
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ZOOLOGICAL SCIENCE 9: 1129-1130 (1992)
[THE ZOOLOGICAL SOCIETY PRIZE]
© 1992 Zoological Society of Japan
GENE EXPRESSION INVOLVED IN MELANOCYTE
DIFFERENTIATION IN THE MOUSE
TAKUJI TAKEUCHI
Biological Institute, Faculty of Science, Tohoku University, Aoba-yama,
Sendai 980, Japan
One of the best-suited cell types for the study of
cell differentiation is the melanocyte for a number
of reasons. 1) It possesses a biochemical marker,
tyrosinase, that is the key enzyme for melanogene-
sis and is the specific protein to the melanocyte. 2)
It possesses a morphological marker, melanosome,
that is the specific organelle to the melanocyte. 3)
Moreover, a number of mutants related to melano-
cyte differentiation and expression have been
found. This means that there exist various gene
loci, each of which controls a step of melanogene-
sis or melanocyte differentiation.
Genes involved in melanocyte differentiation
can be classified into three groups. One consists of
genes that control intercellular communication in
early differerentiation of melanoblasts from neural
crest cells. One of the white spotting genes, W, has
been shown to code for a tyrosine-kinase type
receptor known as c-kit, while the Steel (S/) locus
is demonstrated to be the ligand for the c-kit
receptor. The second category consists of genes
that control proteins specific to the function of the
melanocyte. This group of genes includes the c
locus which encodes tyrosinase and the b locus for
the melanosome protein. The third group consists
of genes that control signal transduction involved
in the hair pattern formation. Genes at the agouti
(a) locus and the extension (e) locus determine the
type of melanin synthesized in the hair follicle
melanocytes, thereby controlling the hair pattern
formation.
In order to elucidate the structure of the
tyrosinase gene (c) and the mechnism of regulation
of gene expression, we cloned the tyrosinase
cDNA and the genomic 5’-flanking sequences.
One of the cDNA cloned was found to consist of
1978 bp. The open reading frame is shown to
encode 533 amno acids with two putative copper
binding sites. We then constructed a minigene in
which the cDNA was ligated with the genomic 5’
flanking sequence of 2.6 kb, and tansfected cul-
tured albino melanocytes with the construct. We
found that the minigene expressed and dierected
the production of melanin pigments in the albino
melanocytes. This result indicates that both the
cDNA and the 5’-flanking sequence are functional.
We also microinjected the minigene into fertil-
ized eggs of albino mice and demonstrated that the
transgenic mice produced melanin pigments. In
order to verify the cell-type specific expression of
the transgene, histological examinations were per-
formed on various organs of the transgenic mice.
Melanin pigments were observed only in hair
bulbs, hair shafts, choroid and pigment epithe-
lium, whereas the transgenes were detected in
various tissues examined by Southern blot analysis
using a 1-kb fragment of the cDNA as the probe.
It seems that the 5’-flanking sequence deriving
from the genomic tyrosinase gene contains cis
elements responsible for the cell typespecific pro-
tein factors even if the transgenes are integrated
randomly among chromosomes.
By crossing the founder mice with albino mice,
transgenic lines and sublines were established.
Each subline expressed a characteristic phenotype
with its respective band patterns in Southern blot
analysis. The difference in phenotypes among
sublines is problably due to the position effect of
the chromatin where the transgene is integrated.
On the other hand, two highly homologous
sequences were found in the 5’-flanking regions of
the c-gene and the b-gene. These two sequences
1130
are designated as p-MSE (10 bp) and d-MSE (13
bp). The gel retardation assay showed that the
mobility of two retarded bands was similar for both
genes. This indicates that at least two different
proteins interact with the 5’-regulatory regions of
both the c-gene and the b gene. South-Western
blotting analysis demonstrated two major proteins
(43 kD and 50kD) that were common to the
regulatory regions of both genes. Therefore, it
seems reasonable to assume that multiple genes
are regulated coordinately to produce melanin in
the melanocyte as a “regulon”.
ZOOLOGICAL SCIENCE 9: 1131 (1992)
[THE ZOOLOGICAL SOCIETY PRIZE]
© 1992 Zoological Society of Japan
STUDIES ON THE HATCHING ENZYME AND ITS SUBSTRATE,
EGG ENVELOPE, OF ORYZIAS LATIPES
KENJIRO YAMAGAMI
Life Science Institute, Sophia University 7-1 Kioicho,
Chiyoda-ku, Tokyo 102, Japan
Enzymatic hatching of fish embryos consists of
may elemental or constitutive processes as follows:
(1) Expression of hatching enzyme gene(s), (2)
Formation and accumulation of hatching enzyme
in association with differentiation and maturation
of hatching gland cells, (3) Secretion of hatching
enzyme, (4) Egg envelope breakdown by the
secreted enzyme, and (5) Emergence of the
embryos. Employing the hatching enzyme of the
fish, Oryzias latipes, as material, we have analyzed
some of these processes. The present paper
surveys the results.
Oryzias latipes hatching enzyme was found to be
an enzyme system consisting of two distinct but
similar Zn-proteases, high choriolytic enzyme
(HCE) and low choriolotic enzyme (LCE). By the
use of polyclonal and monoclonal antibodies
against each of them, cDNAs for HCE and LCE
were cloned from cDNA libraries constructed
from RNAs of Day 3 embryos. Analysis of the
cDNAs showed that they were synthesized as
preproenzymes and the propeptide portions were
N-glycosylated. Examination of the structure
around the active site of both the enzymes strongly
suggests that they belong to astacin (protease)
family. Northern blotting and Western blotting
analyses using their cDNA fragments and anti-
bodies, respectively, as probes revealed concur-
rent expression of their genes, followed by an
immediate translation of their transcripts in Day 2
embryos (stages of lens formation to retinal
pigmentation). Double immunostaining of sec-
tions of the secretory granules in situ or in isolation
with the polyclonal and monoclonal antibody
systems indicated that the proenzymes of HCE and
LCE were colocalized to the same secretory
granules and that HCE was located evenly in the
granules, while LCE was situated at the periphery
of the granules.
On secretion, the proenzymes are probably
activated by some EDTA-sensitive protease(s) to
change into mature forms. Although a close
relation between secretory activity and respiratory
activity of prehatching embryos has long been
known, it seems that intracellular Ca* * eventually
plays a crusical role in the secretion of gland cells,
as Ca* *-ionophore induces the secretion instan-
taneously. The secreted and activated enzymes
attack the egg envelope from the inside. It is
conjectured that the major constituents (ZI-1, 2,
3) of the inner layer of egg envelope of this fish are
synthesized in the form of precursors named
SF-substances in the mother’s liver and that they
are associated to form an envelope around an
oocyte. The inner layer of the complete oocyte
envelope becomes tough on fertilization through
hardening process, which comprises covalent
cross-link formation between the associated sub-
unit proteins. HCE and LCE exert a cooperative
choriolytic action; HCE binds to the hardened
inner layer and swells it remarkably through a
partial proteolysis and subsequent hydration. LCE
which can hardly digest the intact inner layer
solubilizes the HCE-swollen inner layer very
efficiently. Clarification of each of the hatching
processes would provide us with some information
of cellular and molecular mechanisms of reproduc-
tive and developmental phenomena.
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Biochemistry 1133
ACTIVATION OF PROPHENOLOXIDASE IN DROSOPH=
ILA. VI. ANALYSIS OF ACTIVATING SYSTEM.
T. Fukumitsu, K. Fujimoto, K. Masuda,
M. Tanaka, N. Asada, E. Ohnishi.
Biol. Lab., Fac. of Sails 4 Okayama
University of Science, Okayama.
In insect, phenoloxidase in hemolymph
occurs as an inactive proenzyme and is
activated upon bleeding. We have
presented evidence showing that the
activating enzyme ( PPAE ) is a serine
protease.
Nature of the activation reaction has
been analyzed with respect to effects of
salts, pH dependency and other factors.
Kinetic experiments revealed that the PPAE
was rapidly inactivated during the
activation reaction.
ACTIVATION OF PROPHENOLOXIDASE IN DROSOPH-
ILA. VII. CHARACTERIZATION AND ACTIVATION
OF Ay.
K. heGaneton T. Fukumitsu, K. Masuda,
M. Tanaka, N. Asada and E. Ohnishi.
Biol. Lab., Fac. of Silo» Okayama
University of Science, Okayama.
Insect phenoloxidase exists as an
inactive percursor (prophenoloxidase;
proPO) and the proPO is converted to an
active enzyme by an activating system. In
Drosophila, it has been reported that
proPOs are consisted of three A
components: Ai» Ao and A3- We have so far
confirmed the two isoforms: A, and A3.
Procedure for the purification of A, was
improved. It consisted of ammonium sulfate
fractionation, DEAE-cellulose,
chromatofocusing and phenyl Sepharose
column chromatography. Purified Ay
migrated as a single band on SDS-PAGE.
Using the homogeneous samples, properties
of the protein were studied.
ACTIVATION OF PROPHENOLOXIDASE IN DROSOPH-
ILA. VIII. PURIFICATION AND CHARACTERIZAT-
ION OF Az.
K. Masuda, T. Fukumitsu, K. Fujimoto,
M. Tanaka, N. Asada and E. Ohnishi. Biol.
Lab., Fac. of Sci., Okayama University of
Science, Okavama.
Insect phenoloxidase occurs as an
inactive proenzyme (prophenoloxidase;
proPO), which is converted to the active
enzyme by the activating enzyme. In
Drosophila, there are two molecular
species of the proP0O, designated as A, and
Az. They are distinguishable by ammonium
sulfate fractionation and native-PAGE. A3
was purified by ammonium sulfaté
fractionation, Sephacryl S-200, DEAE-
cellulose and hydroxylapatite column
chromatography. The properties of A
including molecular weight, isoelectrié
point, thermostability, pH stability and
substrate specificity of the activated
proPO were studied.
ACTIVATION OF PROPHENOLOXIDASE IN DROSO-
PHILA. IX. ACTIVATION OF A, WITH 2-
PROPANOL.
N.Asada, T.Fukumitsu, K.Fujimoto, K.Masuda,
M.Tanaka and E.Ohnishi. Biol. Lab., Fac. of
Sci., Okayama University of Science,Okayama
In D. melanogaster, A, component of the
prophenoloxidase (proPO) could be activated
with both an endogeneous activating system
(AMM-1) and organic compounds including
alcohols. In the activation of A, with
alcohols, 2-propanol was the mos and
glycerol was the least effective among the
alcohols tested. A, was activated within 2
min after the addition of 2-propanol. Rate
of activation and final yield of the PO
activity depended on the concentration of
2-propanol. When the concentration of 2-
propanol was lowered by dilution, PO
activity decreased gradually. Upon re-
addition of 2-propanol to this diluted
mixture, PO activity re-elevated. Thus the
reversibility of the activation of A, in
response to the alteration of the concent-
ration of 2-propanol could be observed.
The maximum level of the PO activity,
which had been activated with 2-propanol,
was higher than that activated with AMM-1.
Optimum concentration of 2-propanol for the
rate of activation was 50 %.
The activated state of A, showed proper-
ties of a tyrosinase-type. The results
indicate that the activation of A, with 2-
propanol is caused by the reversible con-
formational change of the proPO molecule.
1134 Biochemistry
ACTIVATION OF LACCASE-TYPE PROPHENOLOXIDASE
IN THE CUTICLE OFUENSECT. IK.PROPERTIES OF
PROLACCASE IN LARVAL CUTICLE OF SILKWORM,
BOMBYX MORI. z
H. 1. Yamazaki., Biol., Lab.,Atomi Gakuen
Women’s Univ., Saitama.
The hardening and darkening mechanism of
cuticle has been studied extensively on
larval-pupal ecdysis.The laccase,a type of
phenoloxidase is found in cuticular matrix
and supposed that the enzyme mediates the
hardening and darkening process. And the
laccase itself is involved in the newly
tanned matrix. The laccase is already found
in newly formed pupal cuticle as an
inactive proform (prolaccase) bound with
matrix and it is able to isolate and acti-
vate by proteinase.
Prolaccase was also found in the cuticle
tanning process for larval-larval ecdysis
between 4th and 5th istar. The larval
prolaccase activated by trypsin and the
activation was completely inhibited by
1 M NaCl. The prolaccase could not be
activated by pepsin different from the
case of pupal prolaccase.
The cuticle hardening mechanism is funda-
mentally similar between larval-pupal and
larval-larval ecdysis. However,it was
observed that the peptides involved in the
process were slightly different.
PROPHENOLOXIDASE ACTIVATING SYSTEM IN
CUTICLE OF THE LARVAL SILKWORM, BOMBYX
MORI.
M. Ashida, Y. Koizumi and P. Brey. The
Institute of Low Temperature Science,
Hokkaido Universit Sapporo.
Three types of cuticular enzymes which
are capable of oxidizing phenolic
compounds have been reported in the
literature. They are laccase and two
types of tyrosinase-type phenoloxidase.
One of the phenoloxidases is called injury
phenoloxidase, because it exerts its
action when the cuticle is injured. The
injury phenoloxidase is claimed to be
present as an inactive form (prophenol-
oxidase, proPO) in cuticle, but its
presence remains to be demonstrated.
We studied the regulation mechanism of
phenoloxidase activity in the larval
cuticle of the silkworm, Bombyx mori. We
developed a method to extract the "injury"
Phenoloxidase as an inactive enzyme
(cuticular proPO). The cuticular proPO
was shown to be activated through a
limited proteolysis. As our studies using
protease inhibitor indicated that
cuticular proPO activating enzyme is also
present as an inactive enzyme, cuticular
proPO seemed to be activated by the action
of a cascade.
A method to obtain a homogeneous
cuticular proPO and the immuno-
cytochemical localization of the enzyme in
cuticle were also reported.
PURIFICATION OF PRO-BAEEase (a serine enzyme
zymogen) IN PROPHENOLOXIDASE CASCADE OF INSECT
AND ITS ACTIVATION TO AN ACTIVE FORM.
Y.Katsumi and M.Ashida. Biochemical. Lab.,
The Institute of Low Temp. Science, Hokkaido
University,, Sapporo.
In insect hemolymph prophenoloxidase has been
shown to be activated through the action of a
cascade, which is termed prophenoloxidase cascade.
Although two zymogens of serine protease,
proBAEEase and proPPAE (prophenoloxidase
activating enzyme) are known to be activated
sequentially when the cascade is triggered by
microbial cell wall compornents such as
f-1,3-glucan and peptidoglycan, none of them has
been prified yet.
We developed a method to purify proBAEEase
from hemolymph of the silkworm, Bombyx mori.
The purification procedures consisted of
fractionation by ultracentrifugation and ammonium
sulfate and column chromatography on Sephadex
G-150, Pheny1-Toyopearl, Heparin-Toyopear],
hydroxylapatite, and Mono-Q
The purified proBAEEase migrated as a single
polypeptide with a molecular mass of 39kDa.in SDS
-PAGE. The results of the experiments to
partially reconstruct prophenoloxidase cascade
using the purified proBAEEase and a fraction
obtained during the purification of proBAEEase
were reported
A STUDY ON THE RELATIONSHIP BETWEEN THE
CROSS-LINKING OF INSECT CUTICULAR PROTEINS
AND THE BIOSYNTHESIS OF PAPILIOCHROME II.
Y. Umebachi. Izumino-machi 2-12-24,
Kanazawa.
Papiliochrome II is a pale yellow pig-
ment in the wing-scales of papilionid bu-
tterflies. The structure has been report-
ed to be N® -[%-(3-aminopropionylamino-
methyl)-3,4-dihydroxybenzy1]-L-kynurenine,
in which the aromatic amino nitrogen of
L-kynurenine is bonded to the side chain
8 -carbon of N-f-alanyldopamine (Rembold
and Umebachi, 1984). This binding can be
enzymatically made by the extract of some
insect cuticles or colleterial glands (
Yago, 1989; Sugumaran et al., 1990). On
the- other hand, N-acyldopamines like N-
acetyldopamine and N-§-alanyldopamine are
known to function as cross-linking agents
in insect cuticle (Andersen, 1985; Kramer
and Hopkins, 1987; Sugumaran, 1988).
There are two kinds of cross-linking me-
chanism: (1) quinone-tanning and (2) #-
(or &, B-)sclerotization. In the latter
mechanism, amino or imino nitrogen of pro-
tein is bonded to the side chain @-(or
&, A-)carbon of N-acyldopamine by both
phenol oxidase and quinone-methide isome-
rase (Saul and Sugumaran, 1989). All av-
ailable evidence indicates that both the
enzymatic synthesis of Papiliochrome II
and the @-sclerotization of cuticular pro-
teins come under the same category.
Biochemistry 1135
THE ROLE OF PIGMENT BINDING PROTEIN IN
XANTHOMMATIN BIOSYNTHESIS IN THE EPIDERMAL
CELLS OF THE SILKWORM, BOMBYX MORI.
H. Sawada’, M. Tsusué! ,T. Iino?
‘Biological Lab.School of Liberal Arts, Kitasato Univ.,
Sagamihara Dept. of General Education, Nihon Univ., Tokyo
We have already reported that the pigment protein separated by
SDS-PAGE had an activity binding with xanthommatin and
cinnabarinic acid. However, 3-hydroxy-kynurenine or 3-
hydroxy-anthranilic acid did not bind to the protein. These data
indicate that the protein has an affinity with phenoxazinone ring
of xanthommatin. Biosynthesis of xanthommatin was also
investigated. The homogenate of epidermal tissue was fractionated
by sucrose density gradient centrifugation. When the substrate
3-hydroxy-kynurenine was added to each fraction, catalytic
activity to form xanthommatin localized in the pigment granules
fraction. In addition, HPLC analysis revealed the incorporation
of 3-hydroxy-kynurenine into the intact pigment granules. These
data suggest that the pigment granule has an important role in
both biosynthesis and accumulation of xanthommatin.
Recently, we obtained polyclonal antibody from the pigment
protein which was purified by SDS-PAGE. Furthermore, we
are now investigating the cross-reactivity of the antibody with
crude extract of epidermis of w2 mutant which is unable to
synthesize the pigment.
CHARACTERIZATION OF RAT AND HUMAN SEPIA-
PTERIN REDUCTASE GENES
H.Ichinose, K.Titani, K.Fujitea, T.Nagatsu
(Inst.for Comprehen. Med. Sci., Sch.of Med.,
Fujita Health Univ., Toyoake), and
S.Katoh, T.Sueoka (Dept.of Biochem., Sch. of
Dent., Meikai Univ., Sakado) .
Biologically active biopterins observed
in insects and vertebrates are produced by
the function of sepiapterin reductase (SPR) .
We studied on the characterization otf rat
and human genes of SPR. We have isolated a
full-length cDNA clone for SPR from a human
liver cDNA library by plaque hybridization
and analyzed the nucleotide sequence of the
cDNA’. We amplified the cDNA for rat SPR by
the PCR.Synthetic primers for the amplifica-
tion were designed based on the nucleotide
sequence of rat SPR* and the amino acid
sequence of the mature form of rat SPR*. The
clone encoded a protein of 261 amino acids
(783 bases) with a calculated Mr of 28047
daltons.A single gap of codon was introduced
into the human SPR sequence against rat SPR
[1*AGG] . Consensus sequences for NADPH and
pterin located in the range near the 5'-end
in both SPR. Pterin binding site |GCCGGGTTG-
CTGTCG (A-G-L-L-S)] for rat SPR? was revealed
as |GCCTCGCTGCTGTCG (A-S-L-L-S) ] 1n human SPR.
Estimation of the number of nucleotide sub-
stitution was 0.250+ 0.021 (total;JC method) ,
and 0.262, 0.105, and 0.429 at the 1,2, & 3
base positions of codon, respectively (K3P
method) . Human SPR showed a 74% identity in
amino acid sequence with that of rat SPR.
Rate of amino acid substitution was1.9x10°%.
1Tchinose et al'91,BBRC179,183;7Citron et al
"90,PNAS87,6436;*Oyama et al‘90,BBRC173,627.
PTERIDINES IN THE YELLOW-COLORED CHROMATO-
PHORES OF THE ISOPOD, ARMADILLIDIUM VULGARE.
M.Nakagoshi, S. Takikawa and S. Negishi.* Biol. Labo.,
Kitasato Univ., Sagamihara. *Dept. of Biol., Keio Univ.,
Yokohama.
In A. vulgare the ommochromes in the integument impart a
dark gray or brown body color. The male of A. vulgare displays
a uniform dark gray color, while the female usually displays a
brown color with characteristic yellow markings aligned along
the dorsal region. Principal component of the yellow pigment
isolated from the yellow markings was already identified as
sepiapterin. Morphological investigations revealed that the
pattern of yellow-colored chromatophores in the female was
externally observable at the dorsal surface of the integument as
the yellow markings. In contrast, the yellow-colored
chromatophores were not externally observable in the male,
since they were covered by an ommochrome chromatophore
layer. The yellow-colored chromatophores contained numerous
granules in the cytoplasm and the morphological properties of
the granules were similar to those of pteridine granules which
contain unc acid occurring in the silkworm integument. Based
on TLC, HPLC and UV-spectrophotometric analyses, we
concluded that blue and violet fluorescent compounds isolated
from the chromatophores were biopterin, pterin and
isoxanthopterin. Uric acid also accumulated in the
chromatophores. The content of both sepiapterin and biopterin
in the male was about two times greater than in the female,
while the content of both pterin and isoxanthopterin showed
few difference between the male and female. The quantitative
difference in sepiapterin and biopterin between both sexes
Suggests that the activities of various enzymes involved in
pteridine metabolism may differ between the male and female
of A. vulgare.
THE STUDY ON BODY COLOR OF ARMADILLIDIUM
VULGARE BY THE FINE STRUCTURE OF EPIDERMIS
AND PIGMENT QUANTITY.
S.Negishi! Y.Haseqawa! , Y.Katakura? ,
P.Juchavlt3, G.Martin3. 'Dept.of Biol.,
Keio Univ., Yokohama, ZDept.of Bioengin.,
Soka Univ., Tokyo and “~Lab.of Animal Biol.
Univ.of Poitier, France.
A.vulgare is polymorphic for the body
color. Red body color is dominant over the
usual black or grey (wild type).
Polymorphism may be provided by the
difference in the fine structure of
pigment granules and/or pigment quantity
under the control of proper genes. The
study was undertaken to prove this
hypothesis.
Chromatophores of the red phenotype
were filled with pigment granules of
filamentous structures such as immature
granules + occurring within the same
limiting membrane. Xanthommatin content of
red A.vulgare is much the same with that
of wild type. Epidermis of white A.vulgare
1s very partially pigmented in the large
vesicles. This observation suggests that
white woodlice provide the enzyme for
ommochrome synthesis and its activity may
be prohibited though the mechanism is
uncertain. These results show that the
structure of pigment granules is
determined by the corresponding gene to
the body color.
1136 Biochemistry
A SIMPLE CRITERION FOR PREDICTING
WHETHER OR NOT A MYOGLOBIN HAS THE
USUAL DISTAL HISTIDINE RESIDUE
A. Matsuoka and K. Shikama. Biol. Inst., Tohoku
Univ. Sendai
Myoglobins from various species can be divided
into two groups by an absorbance ratio of the Soret
peak of the acidic met-form to that of the oxy-form,
namely the Ymet / Yoxy ratio.
Values higher than 1.0 (ranging from 1.16 to
1.41) were thus obtained for the myoglobins
containing the usual distal histidine, whereas those of
less than 1.0 (ranging from 0.79 to 0.84) were the
ratio for the myoglobins lacking this residue, such as
those from three kinds of gastropodic sea molluscs
and two kinds of sharks.
On the basis of these Soret absorption spectra,
we have also examined the unique structures of a
protozoan myoglobin from Paramecium caudatum,
an annelid giant hemoglobin from Tylorrhynchus
heterochaetus, and an insect hemoglobin from
Tokunagayusurika akamusi.
Shikama, K. and Matsuoka, A. (1989) J. Mol.
Biol. 209, 489-491.
ELEPHANT MYOGLOBIN WITH THE DISTAL
GLUTAMINE : AN UNUSUAL STABILITY
PROPERTY OF OXYMYOGLOBIN
T. Tada, A. Matsuoka and K. Shikama. Biol.
Inst., Fac. of Sci., Tohoku Univ., Sendai.
In the usual mammalian myoglobins, the distal
(E7) histidine 1s known to play a key role in the
stability properties of the bound dioxygen.
Elephant myoglobin, however, lacks this residue and
has a glutamine at E7 position.
In order to know the effect of the distal residue
on the stability, we have isolated native
oxymyoglobin (MbO2) directly from the cardiac and
skeletal muscle tissues of the African elephant
(Loxodonta africana ), and examined the
autoxidation rate from MbO?2 to metMb over the
wide range of pH 4.5 - 12.3 in 0.1 M buffer at 25°C.
The pH profile obtained was similar to that of
Aplysia MbO2 bearing the distal valine, but elephant
MbOz2 was found to be less susceptible to
autoxidation and its extent was almost comparable
with sperm whale MbO3.
ROLE OF THE DISTAL RESIDUE ON THE
REACTION OF METMYOGLOBIN WITH
HYDROGEN PEROXIDE : A COMPARATIVE STUDY
G. Tajima', A. Matsuoka and K. Shikama’.
‘Dept. of Biol. Sci., Coll. of Gen. Edu., and “Biol. Inst.,
Fac. of Sci., Tohoku Univ., Sendai.
Metmyoglobin reacts with hydrogen peroxide to
form ferryl-myoglobin, which can revert back
spontaneously to the met-form. This reaction seems to be
of physiological importance, since through this cyclic
reaction of myoglobin between metMD(III) and ferryl-
MDb(IV), H,O., one of the most potent oxidant in vivo,
can be decomposed continuously in red muscle tissues in
the absence of catalase and peroxidase (Tajima, G. and
Shikama, K. (1992) Int. J. Biochem. in press .).
We have isolated native metmyoglobins from various
species, and examined the mode of reaction with
hydrogen peroxide in relation to the kinds of the distal
(E7) residue. The spectroscopic results have shown that
metmyoglobins lacking the usual distal histidine, such as
those from African elephant, shark (Galeus nipponensis),
and three species of gastropoda, Aplysia kurodai, Aplysia
juliana and Dolabella auricularia, are little or never
converted to ferryl-form.
EFFECT OF PHOTOIRRDIATED TIN-PROTOPOR
PHYRIN ON ARYLSULFATASE ACTIVITY OF RAT
BRAIN LYSOSOMES.
H. Keino, and T. Banno. Dept. Perinatol.,
Inst. Dev. Res., Kasugai.
"A synthetic heme analogue (tin-protopor
phyrin; SnPP) is known to cause serious
responses of human babies and suckling
rats to photoirradiation. Le alisio
suggested that photo-excited porphyrins
cause great damage to lysosomal membranes.
In this study we report the effects of
SnPP plus photoirradiation on a lysosomal
enzyme (arylsulfatase; ASase). The crude
mitochondria fraction was obtained from
the brain of suckling rats. The mitochon-
dria-free lysosomes were then isolated
from the crude mitochondria fraction by
Percoll density gradient centrifugation.
ASase activity was markedly reduced by
photo-excited SnPP. No reduction of ASase
activity was detected under the dark.
Photoirradiation never reduces the ASase
activity without SnPP. The reduction was
prevented by administration of L-ascorbic
acid and was reinforced by D920. Photo-
excited SnPP may bring singlet oxygen and
inaugurate oxygen-free radical reactions.
The kinetic study demonstrates that
apparent Km value of ASase for 4-nitro-
catecholsulfate was calculated to be
0.39mM in both presence and absence of the
photo-excited SnPP. Photo-excited SnPP
reduced the velocity of ASase activity.
Biochemistry 1137
METMYOGLOBIN AUTOXIDATION AND REDUCTION
M.Kariya!, K.Machida!, H.Namiki?.
1Dept. of Sports Sci., Sch. of Human Sci.,
Waseda Univ., Tokorozawa, 2Dept. of
Biol., Sch. of Educ., Waseda Univ., Tokyo.
In muscles such as the cardiac and the
skeltal, myoglobin (Mb) plays a role in
maintaining aerobic metabolism, both as an
oxygen store and by facilitating oxygen
diffusion. The reduced form of Mb is
required for this reversible oxygenation
to occur. However, myoglobin is easily
transformed into metmyoglobin (metMb) by
autoxidation. Muscles therefore contain
metMb reductase. We presently report a
reducing factor in beef heart muscle dif-
ferent from the known metMb reductases.
The factor was separated between m.w.
1000 and 3000, by ultrafiltrations. The
crude sample was then applied to a HPLC
column of anion exchanger and frac-
tionated. A metMb reducing activity was
separated in one fraction. This factor
reduced metMb faster with NADPH than with
NADH. Some other hemoproteins were as-
sayed if they served as electron ac-
ceptors. Methemoglobin was hardly
reduced, but cytochrome c was reduced. It
also reduced an artificial dye 2,6-
dichlorophenolindophenol. The pH optimum
of this NADPH-diaphorase activity is about
6.0.
PHOTO-ACTIVATION OF RESPIRATION IN ABALONE
SPERM.
E. Tazawa', A. Fujiwara? and I. Yasumasu?
‘Biol. HASTE 5 p Yokohama City Unilvin;
Yokohama and ?Dept. of Biol., School cf
Education, Waseda Univ., Tokyo.
In sperm of the abalone, Norditis discus
the respiratory rate was enhanced by light
irradiation with the peaks of activating
effect of light at 430, 550 and 570 nm.
The wavelengths at the peaks of activating
effect on respiration correspond to those
at peaks of light absorption of cytochrome
be Probably, photo-activation of
cytochrome b results in augmentation of
respiration in abalone sperm. In sperm of
sea urchin, starfish and echiuroid, photo-
activation of respiration with peaks
corresponding to the absorption peaks of
cytochrome b was not found, unless eperm
were exposed to PMS or DCPIP.
Acceleration of electron supply to
cytochrome b by these compounds probably
makes photo-activation of cytochrome b
reaction apparent as an increase in the
respiratory rate in these sperm. This
study was carried out under the NIBB
(National Institute for Basic Biology)
Cooparative Research Program for the
Okazaki Large Spectrograph (91-502) .
HIGH LEVELS OF AL,FE AND MINOR ELEMENTS BOUND TO
SKELETAL PIGMENTS IN A CORAL OULASTREA CRISPATA
H. Yamashiro. Radioisotope Lab., Univ. of the Ryukyus, Okinawa
A zebra coral Oulastrea crispata
(Faviidae Scleractinia) has colored
skeleton (gray to black). Skeletal
Pigments are separated from the skeleton
by the treatment of weak acid. Resultant
acid-insoluble matter containing Fe has
been reported (not quantitative study).
The present study was done to measure the
amount of Fe and other minor elements
bound to skeletal pigments by neutron
activation analysis. Highly concentrated
Fe (>9500ppm) and Al (>14000ppm) were
detected in the acid-insoluble matter of
the skeleton. Furthermore, this matter
contained high levels of minor elements
such as Sc, V, Cr, Br, Ag, I, La, Ce, Sm,
Hf, Th and U with the value more than
2ppm. Concentration factors reached x10 7
for Al, Ag, Ce and Sm. These results
suggest that the acid-insoluble organic
substance of the coral skeleton has a
powerful affinity for Al, Fe and many
other minor elements. Coral soft tissues
seem to be involved in the concentration
of these elements either at the time of
uptake or calcification.
DIABETIC PERIPHERAL NEUROPATHY AND
DECREASE IN MICROTUBULE-ASSOCIATED PROTEIN
KINASE ACTIVITY.
T.Kobayashi , K.HouiZ, Y.Mori2 ,H.Sasaki2
gna SMO ChAC mr ‘Dept. of Biochem. and
the 3rd Dept. of Intern. Med., Jikei
Univ. School of Med., Tokyo
The activity of microtubule-associated
protein (MAP) kinase is controlled through
phosphorylation of its tyrosine residue by
either of insulin receptor or nerve growth
factor receptor, and the enzyme is regard-
ed to play important roles in cell prolif-
eration and neuronal functions. We consid-
er that decreased insulin fails to stimu-
late MAP kinase activity to the sufficient
level even in the presence of nerve growth
factor (NGF) and induce diabetic neuropa-
thy. We investigated MAP kinase activity
in a new insulin-defective strain of rat
(WBN/Kob) and found that the activities
were remarkably decreased in every neural
tissues of the animals at the age of 15
months. Especially, the activity in sciat-
ic nerve was only 19% of that of the
control. On the other hand, the activities
in spleen and in submandibular gland were
elevated to 400% and 250%, respectively.
We then employed cultured dorsal root
ganglion cells isolated from 10 day chick
embryo. When they were cultured in the
absence of insulin, their neurite elonga-
tion, MAP kinase activity and contents of
MAP1 and MAP2 were all decreased remarka-
bly.
1138 Biochemistry
ANALYSIS OF THE PROCESSING OF ALZHEIMER
AMYLOID PROTEIN PRECURSOR EXPRESSED IN
COS CELLS BY cDNA TRANSFECTION.
Wakako Yamao-Harigayal,2, Mihoko Usamil and
Kei Maruyama!,2. 1Department of Molecular Biology,
Tokyo Institute of Psychiatry, Kamikitazawa,
Setagaya, Tokyo 156, and 2Department of Molecular
Biology, Tokyo Metropolitan Institute of Clinical
Medicine, Honkomagome, Bunkvo, Tokyo 113.
One of the pathological features of Alzheimer's
disease (AD) is the deposition of 8/A4 protein (%P) as
senile plaque. BP is a 42-amino-acid protein and it is
derived from a 695-amino-acid precursor, amyloid
precursor protein (APP). In normal condition, APP is
cleaved in the interior of 8P by the hypothetical
protease “secretase,” thus preventing its production.
The cause of neuronal degeneration and AD might be
the abnormal processing of APP. Since the expression
of APP is fairly low, APP was expressed transiently in
COS-1 cells by cDNA transfection to study its
processing. APP695 and APP770 (with Kunitz-type
protease-inhibitor domain) were processed in a similar
way to produce 100 kd and 9 kd fragments. Three
kinds of mutations of APP, Val717-Ile, Phe or Gly,
were reported in familial AD. These mutations had no
effect on the processing of APPs expressed in COS-1
cells. This means that these mutations might not be a
direct cause of AD. The mutations of the proposed
cleavage site of secretase had no effect. Hence
secretase seemed to recognize the conformation of
APP rather than its amino-acid sequence. The
identification of secretase is now under way.
THE INHIBITORY MECHANISM TO TRYPSIN OF A
58 KDA INHIBITOR FROM THE HEMOLYMPH PLASMA
OF HALOCYNTHIA RORETZI.
T. Abe, F. Shishikura, S. Ohtake and K.
Tanaka. Dept. of Biol., Nihon Univ. Sch.
of Med., Tokyo.
A 58 kDa plasma inhibitor inhibited
bovine pancreatic trypsin as well as its
endogenous enzyme(s) in the hemolymph of
an ascidian, H. roretzi. To analyze the
inhibitory mechanism of the inhibitor,
bovine pancreatic trypsin was used. The
trypsin activity was first decreased by
the addition of inhibitor in both dose-
dependent and time-dependent Manners,
whereas after prolonged incubation (over
20 hours) the trypsin activity recovered
almost the initial activity of trypsin. On
SDS-PAGE analysis the inhibitor (58 kDa)
associated with the trypsin (23 kDa) to
form a complex (approx. 80 kDa) in reduced
and unreduced samples. This complex re-
sisted to treatment of SDS but easily
dissociated by treatment of a nucleophilic
reagent, such as ammonia. These results
indicated that the complex was covalently
formed by the reaction of the inhibitor
and the trypsin and then the inhibitor
might be degraded by trypsin.
As a similar time course of the inhibi-
tion was found in the enzymatic activity
of the hemolymph of H. roretzi, this
mechanism seems to exist in the inhibito-
ry reaction between the inhibitor and
cognate plasma enzyme(s).
DEXTRAN SULFATE ACCELERATES THE INHIBITORY
ACTIVITY OF PLASMA-ENZYME INHIBITOR FROM
HALOCYNTHIA RORETZI HEMOLYMPH.
F. Shishikura, T. Abe, S. Ohtake and K.
Tanaka. Dept. of Biol., Nihon Univ. Sch.
of Med., Tokyo.
We have tested the abilities of dextran
sulfates (DSs; 500 kDa, 50 kDa, 15 kDa,
and 5 kDa) and other polyanions including
four kinds of glycosaminoglycans (GAGs) to
increase the inhibition of plasma-enzyme
activity by its endogenous inhibitor
purified from the hemolymph of ascidian,
H. roretzi. DS whose molecular weight is
about 50,°500, 15, or 5 kDa (am@ondengos
decreasing activity) dramatically accel-
erated the rate of reaction between plasma
enzyme and its inhibitor in 50 mM Tris-
HCl, pH 8.0, containing 0.5 M NaCl (0.5 M
NaCl-Tris buffer). When each GAG mixed
with the inhibitor in 0.15 M NaCl-Tris
buffer, not only four kinds of DSs but
also heparin, chondroitin sulfate B, and
fucoidan activated the inhibitor more than
those in 0.5 M NaCl-Tris buffer. However,
chondroitin sulfate A, C and phosvitin in
neither 0.5 M NaCl-Tris buffer nor 0.15 M
NaCl-Tris buffer were not effective at
concentrations of 1 mg/ml. In the
presence of 50 kDa DS, plasma enzyme seems
to undergo instantaneous ternary-complex
formation with inhibitor and DS. Poly-
brene (10 pg/ml) abolished the abilities
of GAGs. These results suggest a similar
mechanism proposed in the activation of
antithrombin III in mammalian blood.
PRIMARY STRUCTURE OF SP-25, A SPECIFIC
SUBSTRATE FOR ATP-DEPENDENT PROTEASE IN
BOVINE ADRENAL CORTEX, AND ITS COMPARISON
WITH MER; 5 IN MOUS ERYTHROL UKEMIA
S.Watabe!, T.Hiroi H Kohno K. .Akiyama;,
H.Kouyama 4 S. Tatunami}, N. Yago 5 Hts Hara;,
-Ohnishi T.Suzuki~ and [.Nakazawa
Radioisotope Res. Inst. and Ist Dept. of
Anatomy, St. Marianna Univ.,Kawasaki, and
Dept.of Biol. Toho Univ. Funabashi.
We purified SP-25, a specific substrate
protein for ATP-dependent protease in
bovine adrenocortical mitochondria and
determined its amino acid sequence using a
protein sequencer after fragmentation and
purification of the fragments. By homology
search using NBRF data base it was found
that SP-25 had 92 % homology with an amino
acid sequence coded by a cDNA clone, MER-
5, in mouse erythroleukemia cells. We iso-
lated a cDNA clone coding bovine SP-25
using a PCR-amplified cDNA fragmant as a
probe. The amino acid sequence deduced
from the nucleotide sequence contained a
presequence specific to mitochondrial
proteins, which had less homology with
MER-5 than mature protein region had.
Since it was reported that MER-5 increased
transiently after induction of differen-
tiation in erythroleukemia cells, and that
removal of MER-5 mRNA by an antisence RNA
inhibited the differention, MER-5 and SP-
25 might have an important role in cell
differentiation.
Biochemistry 1139
ISOLATION AND CHARACTERIZATION OF
PHOSPHOLYPASE A2-LIKE PROTEIN FROM
BULLFROG PITUITARY GLAND.
H.Hayashi!, M.Sakai?, H.Takasu?, S.Tanaka!,
Y.Hanaoka! and S.Kikuyama2. !Inst. of Endocrinol.,
Gunma Univ., Maebashi and 2Dept. of Biol., Sch. of
Educ., Waseda Univ., Tokyo.
During the course of isolation and purification of
thyrotropin from the adult pituitary glands of bull-
frog, Rana catesbeiana, a protein that behaved with
thyrotropin until final purification step was found.
This protein was finally separated from thyrotropin
by hydroxyappatite column chromatography. The
molecular mass of this protein was estimated to be
14kDa by SDS gel electrophoresis. The N-terminal
40 residues and some lysylendopeptidase peptides
were sequenced. The sequence of this protein has
about 40% identity with that of phospholypase A2
from Indian cobra. The antibody against this protein
was raised in a rabbit. This antibody did not rec-
ognize bullfrog lutropin, follitropin, thyrotropin and
their subunits. The contents of this phospholypase
A2-like protein in circulating blood of various stages
during bullfrog metamorphosis were measured by
radioimmunoassay. The plasma level of this protein
was low (<15ng/ml) in premetamorphic stages, then
rapidly increased at the onset of climax (40ng/ml),
and declined at the end of metamorphosis. Immu-
nohistrochemical studies using this antibody
showed that this protein is synthesized in glyco-
protein-producing cells in the bullfrog pars distalis.
PURIFICATION AND CHARACTERIZATION
OF ACID PROTEINASE (ENZYME IV) FROM
BULLFROG GASTRIC MUCOSA
T. Inokuchi, K. Kobayashi and S. Horiuchi.
Life Sci. Inst., Sophia Univ., Tokyo.
Enzyme VI is acid proteinase and exists in both
larval fore-gut and adult stomach of bullfrog, Rana
catesbeiana. This enzyme was purified from adult
gastric mucosa by Hydroxyapatite fast flow type, @
Sepharose fast flow, Con A-Sepharose and Mono
Q HR column chromatography. Enzyme IV was
divided into four peaks (IV-1, IV-2, IV-3 and IV-4) by
Mono Q HR. Mobilities of the four enzymes on
PAGE were slightly different from each other. M.W.
of IV-2 and IV-3 was estimated to be 50,000 in
reducing condition but about two times higher in
non-reducing condition by SDS-PAGE. By acid
treatment their M.W. decreased. The result showed
conversion of pro-enzyme to its activated form,
similar to conversion of pepsinogen to pepsin at
acidic pH. Enzyme VI-2 hydrolyzed bovine
hemoglobin with maximum activity between pH 2-3
but it showed lower activity on BSA, y -globulin or
casein than on bovine hemoglobin. From these
results we thought that enzyme IV was cathepsin E-
type enzyme in anuran.
ACTIVATION OF CYSTEINE PROTEINASE OF THE
SILKMOTH, BOMBYX MORI.
S. Y. Takahashi and Y. Yamamoto. Dept.
of Biol., Fac. Liberal Arts, Yamaguchi
Univ., Yamaguchi.
Cysteine proteinase purified from the
silkmoth, Bombyx mori, can be activated
during incubation in the acidic medium
(DH aah Sn. Activation requires the
presence of substrates in the incubation
medium and is inhibited by E-64, specific
inhibitor for cysteine proteinases.
Concomitant with the activation, the
purified enzyme (47 kDa) is converted to
an intermediate (44 kDa), and finally an
active form (39 kDa) which is relatively
stable. These results indicate that
partial proteolysis of the enzyme is
likely to be involved in the activation
process.
The same pattern changes occur in
intact eggs at the beginning of
embryogenesis and the activated form of
the enzyme can be detected throughout the
embryonic life. These facts strongly
suggest that cysteine proteinase is
stored in the eggs as a proenzyme which
is latent and is activated by partial
proteolysis during embryogenesis.
The regulatory mechanism of the
cysteine proteinase and involvement of
the proteinase in the yolk protein
degradation will be discussed in relation
to the embryogenesis of the silkmoth,
Bombyx mori.
BIOSYNTHESIS OF THE BLOOD GROUP P ANTIGEN-LIKE
GalNAc#173Gal#174G1cNAc/Glc STRUCTURE: A NOVEL
N-ACETYLGALACTOSAMINYLTRANSFERASE ACTIVITY
IN HUMAN BLOOD PLASMA
A. Takeya’, 0. Hosomi’, N. Shimoda’ and S. Yazawa’
Dept. of Legal Med., Gunma Univ. Sch. of Med.,
Maebashi and ?Japan Immunoresearch Laboratories,
Takasaki.
Human blood group 0 plasma was found to contain
a GalNAc-transferase which catalyzes the transfer
of GalNAc from UDP-GalNAc to Galfl>4Glc, Galfl->
4GlcNAc, asialo-«a,-acid glycoprotein and Galfl-
4G1cNAc#173Gal £124Glc-ceramide, but not to Gal?#1->
3G1cNAc. The enzyme required Mn’* for its activity
and showed a pH optimum at 7.0. The reaction
products were readily hydrolyzed by §-N-acetyl-
hexosaminidase and released GalNAc. Apparent Ka
values for UDP-GalNAc, Mn?*, lactose, N-acetyl—
lactosamine, and terminal N-acetyllactosaminyl
residues of asialo-«,-acid glycoprotein were 0.64,
0.28, 69, 20, and 1.5mM, respectively. Studies on
acceptor substrate competition indicated that all
the acceptors mentioned above compete for one
enzyme, whereas the enzyme is distinguished from
an NeuAc«233Gal -],4-GalNAc-transferase, which
also occurs in human plasma. The methylation study
of the product formed by the transfer of GalNAc to
lactose revealed that GalNAc had been transferred
to the carbon-3 position of the 8-Gal residue.
Although the GalNAc£173Gal structure is known to
have the blood group P antigen activity, human
plasma showed no detectable activity of Galel74Gal
§-1,3-GalNAc-transferase, which is involved in the
synthesis of the major P antigen, GalNAc#173Gale]-
4Gal#1+4Glc-ceramide. Hence, the GalNAc#173Gal 61>
4GlcNAc/Gle structure is synthesized by the novel
Galf1>4GlcNAc/Gle -1,3-GalNAc-transferase.
1140 Biochemistry
A MODIFIED METHOD TO OBTAIN A STABLE
TREHALASE FROM ARTEMIA SALINA.
Z. Nambu and F. Akiyama-Nambu, Dept. of
Biol., Univ. of Occupational and
Environm. Health, Japan, Sch. of Nursing
and Med. Technol. Kitakyushu.
As we reported previously, the
trehalase from the nauplii of Artemia
salina specifically hydrolyzes &d-
trehalose. The activity was found to be
inhibited by $-D-glucosides and so we
tried to purify the enzyme by introducing
p-aminophenyl g-D-glucoside - Sepharose
chromatography into our isolation
procedure. The enzyme was purified by
acetone treatment, DEAE-Sepharose CL-6B,
Con A-Sepharose, p-aminophenylppeglucoside-
Sepharose, Poly(A)-Sepharose and gel
filtration using HPLC with a MTSKgel
G3000SW column. The activity of the
trehalase was adsorbed on the
p-aminophenyl 9Q-D-glucoside — Sepharose
column with O.5 mM piperazine-HCl (pH 6.5)
and was eluted by 100 mM piperazine-HCl
(pH 6.5). The yield of the activity was
1.4 % and the specific activity was 17.7
units/mg protein. The obtained enzyme was
shown to be stable in a phosphate buffer
pH 7.0 at 4°C for 10 days and the
stability was prolonged for 26 days by a
treatment of the enzyme with 1 mM
dithiothreitol. The p-aminophenyl
§-D-glucoside-Sepharose chromatography
seems to be effective to some extent for
the preparation of the enzyme.
A MUSCLE ACTIN GENE FAMILY IN THE ASCIDIAN
HALOCYNTHIA RORETZI.
T. Kusakabe!, K. W. Makabe?, N. Satoh’.
1Dept. of Zool., Fac. of Sci., Kyoto Univ., Kyoto.
?California Institute of Technology, Pasadena, USA.
Our previous studies have shown that at least five
muscle-type actin genes form a cluster in the Halocynthia
roretzi genome. Some of these genes are specifically
expressed in larval muscle cells. In this study, we isolated
another muscle-type actin gene, HrMA1 from an H. roretzi
genomic library. The HrMA1 gene was fairly different in the
nucleotide sequence and structure from actin genes in the
cluster Northern blot analysis showed that HIMA1 mRNA
was undetectable in zygotes and cleavage stage embryos.
HrMA1 mRNA first appeared at gastrula stage and
accumulated during later embryogenesis. However, no
HrMA1 transcript was observed in adult tissues including
body-wall and heart muscles. That is, the expression pattern
of HrMA1 was the same as those of actin genes in the cluster
A coding region probe derived from an HrMA2 cDNA
detected transcripts in adult body-wall and heart muscles but
not in non-muscle tissues. Furthermore, the length of
transcripts was slightly different between body-walls and
hearts. These results suggest that different muscle-type actin
genes are expressed in the two different types of ascidian
adult muscle.
GENOMIC STRUCTURE OF 29 kDa PROTEIN FROM
THE ASCIDIAN Halocynthia roretzi.
A. Nakamura! and T. Takagi?. !Dept. Pharmacol., Gunma
Univ., School of Medicine, Maebashi and Biol. Inst., Fac.
Sci., Tohoku Univ., Sendai
The body wall muscle of the ascidian Halocynthia roreizi
contains a large amount of 29 kDa protein (HR-29), whose
the function is still unknown. To elucidate the function and
origin of this protein, we have determined the cDNA and
genomic structures. The cDNA was composed of 871 bp
and encoded 251 amino acid residues. The N-terminal 151
residues contained three homologous repeated sequences
and showed no significant homology with other proteins.
On the other hand, the C-terminal 100 residues showed
homology with small heat shock proteins and alpha-
crystallin. Thus HR-29 is composed of two domains. We
obtained two genomic clones (HR-29G1, HR-29G2)
amplified by PCR. HR-29G2 was slightly different from
HR-29 by partial sequence analysis and not analyzed
further. HR-29G1 was exactly same as HR-29 and has
three introns (I, II, III). The largest intron II (1093 bp)
was placed between two domains and included a possible
promoter site and the PELHAM BOX-like structure which
is known to be important for the expression of heat shock
protein gene. Therefore the C-terminal domain is obviously
derived from heat shock protein. Although the origin of N-
terminal domain is not clear, HR-29 is a fusion protein of
two different origins.
MOLECULAR CHARACTERIZATION AND DIFFEREN-
TIAL GENE EXPRESSION OF ASCIDIAN EMBRYONIC
NUCLEAR ANTIGEN HGV2.
S. Fujiwara. Dept. of Biol. Fac. of Sci.
Kochi Univ., Kochi.
We have previously obtained a hybridoma
clone which produces the antibody against
the antigen, named Hgv2, specific to the
nuclei of the oocytes, embryos and larvae
of the ascidian Halocynthia roretzi.
In the present study, a complete nucle-
otide sequence of a cDNA clone encoding
the Hgv2 antigen was determined. The
amino acid sequence deduced from the cDNA
was Similar to amphibian histone-binding
protein Nl, which is thought to be in-
volved in the chromatin assembly process.
Close relation between these two proteins
was confirmed by their similarity in the
amino acid composition and hydropathy pro-
file. Although putative histone-binding
domains, which are two acidic residue-rich
clusters, do not seem to be conserved,
there evidently are two highly acidic
regions in the Hgv2 polypeptide. Hgv2
protein may function as a nucleosome as-
sembly factor during rapid embryonic cell
divisions.
While the Hgv2 protein was constantly
detected in the nuclei of the embryos and
larvae, the amount of the Hgv2 mRNA gradu-
ally decreased during embryogenesis.
Among the adult tissues, the mRNA was
detected only in the branchial sac, except
for the gonad which contains oocytes.
Biochemistry 1141
TISSUE SPECIFICITY OF ARTHROPOD TROPOMYOSIN
J. Miyazaki, K. Yahata, Y. Fujiwara, and T.
Hirabayashi. Inst. of Biol. Sci., Univ. of
Tsukuba, Tsukuba.
Tropomyosin is a protein family composed
of many isoforms which were classified into
two groups, high-molecular-weight and low-
molecular-weight isoforms. We have inves-—
tigated tissue specificity of tropomyosin
in arthropods. Crustaceans had many tropo-
myosin isoforms distributed in a tissue-—
specific manner including low-molecular-
weight isoforms in non-muscle tissues.
Crustacean hearts had isoforms strictly
specific to cardiac muscle. However,
horseshoe crab tropomyosin showed no tissue
specificity, but had low-molecular-weight
isoforms in non-muscle tissues. Therefore,
different results were obtained between
representatives of two groups in arthro-
pods. This prompted us to further investi-
gate tissue specificity of tropomyosin in
other arthropods. In this study, we used
the beetle, centipede, and scorpion which
belong to three different groups in arthro-
pods and obtained the results as follows.
1) Tissue specificity of high-molecular-
weight isoforms was recognized in all the
examined arthropods except the horseshoe
Crab. 2) Low-molecular-weight isoforms
were found in non-muscle tissues in all the
arthropods except the centipede in which
non-muscle tissue was not examined. 3)
Only crustaceans had cardiac-specific
isoforms.
eDNA CLONING OF BOMBYX LECTIN GENE AND
ANALYSIS oH ITS EXPRESSION.
K. Amanai-_, Suzuki-, S. Sakurai2,
T. Ohtaki2. INatl. Inst. for Basic Biol.,
Gkazalct, Univ. of Kanazawa, Kanazawa,
Univ. of Nishougakusha, Shounan.
We have reported previously that the
hemocyte is the major source of lectin in
the hemolmph of the silkworm, Bombyx mori.
In whole extracts of hemocytes from 5th
instar larvae, 50 kDa protein which react
with anti-hemolymph 350 kDa lectin
monoclonal antibody have been detected.
This protein exhibited the same
biochemical properties as the hemolymph
350 kDa lectin. In order to reveal the
function and structure of the hemolymph
lectin and other lectins in various
tissues, cDNA expression library of
hemocytes was constructed and, from this
library, Bombyx lectin cDNA clones were
isolated using monoclonal antibody as a
probe. Northern blotting analysis
revealed that transcripts with an estimate
size of 2.0 kb are expressed in hemocyte,
Ovary and testis, and not in fat body.
Furthermore, the same transcripts are
expressed maternally in the early embryo.
STRUCTURE AND EXPRESSION OF VITELLOGENIN
GENE OF BOMBYX MORI
K.Yano, M.Toriyama, S.Izumi and S.Tomino
Dept. Biol. Tokyo Metropol. Univ., Tokyo
Vitellogenin (Vg), a precursor of major
yolk protein of Bombyx mori is a tetramer
composed of each two molecules of non-
identical subunits. We have cloned the mRNA
and gene sequences for Vg and analyzed
their structures. The Vg gene is composed
of 7 exons interspersed by 6 introns, which
encodes the 5.7 kb Vg mRNA. The primary
structure of peptide deduced from the mRNA
sequence comprised 1782 amino acid
residues with molecular weight of 203.0k.
Since amino-terminal primary structures of
each Vg subunit are mapped on the deduced
amino acid sequence, it is highly probable
that two subunits of the B. mori Vg are
derived from a single precursor peptide by
cleavage. In microsomal fraction prepared
from the female fat body, a putative
precursor peptide with molecular weight of
about 200k was detected by immunoblotting.
Northern hybridization of the fat body
RNA during development proved that the
level of Vg mRNA sharply rises in female
fat body at about a period of larval-pupal
ecdysis. Developmental profile of the
amount of Vg mRNA was consistent with that
of primary transcript of the Vg gene.
These results conclude that the Vg
synthesis in the B. mori fat body is
regulated in a sex- and stage-dependent
Manner at the level of transcription.
CONSTRUCTION OF cDNA LIBRARY FROM
CRAYFISH PROCAMBARUS CLARKII.
W.-K. Kang and Y. Naya. Suntory Institute for
Bioorganic Research (SUNBOR), Osaka.
Using oligo-dT column, the polyA-mRNAs
were purified from different organs and tissues of
several tens of crayfish. Three to five wg of mRNAs
from four kinds of materials (muscle, liver, eyestalk
and Y-organ) were used to synthesize cDNA by
reverse transcriptase. Then, each cDNA was ligated
to AZAPII vector and used to be packaged. The
average length of inserts from each library was
found to be about 1 kb after PCR (Polymerase Chain
Reaction) that was carried out with SK and T7
primers (of pBluescript vector) and phage stocks of
plaques as templates. By nucleotide sequencing, it
was found to contain ribosomal protein S14
equivalent and actin-homolog in the library of
muscle. Both clones show polyA signal (AATAAA)
and polyA tail in their C-terminal nontranslated
region. The clone 25-3, ribosomal protein S14
equivalent, contains an ORF (151 amino acids) and
shows 88% identity in amino acid level and 77.3%
identity in DNA level to the gene for ribosomal
protein S14 of Drosophila. Actin-homolog clone, 25-
12, is lacking of N-terminal 150 bp, however, shows
87% and 78.2% identity to actin gene of shrimp in
amino acid level and DNA level, respectively.
1142 Biochemistry
PHOSPHORYLATION OF HISTONE H1 DURING DNA
SYNTHESIS IN RAT HEPATOCYTES
K.Asami, .Miyashita, N.Sawada and
T.Kishimoto“. Dept. Biol. and Dept.
Pathol., Sapporo Medical College, Sapporo
060, Lab. Cell and Develop. Biolyn,
Tokyo Inst. Technol., Yokohama 227.
Phosphorylation of histone Hl occurs at
the time of DNA synthesis in primary
cultured rat hepatocytes. Effects of the
compounds which inhibited DNA synthesis in
different ways were examined on
phosphorylation of histone H1 in the
hepatocytes. Sodium butyrate inhibiting
progression of the cell cycle at Gl phase
inhibited both DNA synthesis and histone
Hil phosphorylation. Hydroxyurea and
aphidicolin inhibit DNA synthesis at the S
phase ina different way. They inhibited
both DNA synthesis and H1 phosphorylation
at a similar concentration. But removal of
these compounds from the culture medium
restored DNA synthesis in a little
different way. Removal of hydroxyurea
partially restored both DNA synthesis and
the phosphorylation, while recovery of DNA
synthesis after removal of aphidicolin was
slow and phosphorylation of histone H1 was
very low. Thus, phosphorylation of histone
Hl seemed to proceed as DNA _ synthesis
proceeds. To investigate the protein
kinase responsible for histone Hl
phosphorylation, histone H1 kinase
activity of the cell extract was examined.
The kinase activity increased at the start
of DNA synthesis, and a _ part of the
activity was bound to p13suel beads.
COLD ADAPTATION IN DROSOPHILA
QUALITAIVE CHANGES OF TRIACYLGLYCEROLS
C. Katagiri’, and T. Ohtsu’.
1Biochem. Lab., Inst. of Low Temp. Sci.,
and “Dept. Zool., Fac. Sci., Hokkaido
Univ., Sapporo.
Triacylglycerols are the major fuel for
basal metabolism during the winter in
temperate species of the Drosophila
melanogaster species group. Differential
scanning calorimetry analysis revealed
tuniantey thie staal nis) 14 aso neetemplenacdl tUaEeTS a Ont
triacylglycerol were lower in species or
strains adapted to cooler climate than
those adapted to warmer climates. This
phenomenon was correlated to the fatty
acid compositions of the triacylglycerols;
the proportion of unsaturated fatty acids
in triacylglycerols was higher in the
species or strains adapted to cooler
climates. Furthermore, in the temperate
species of the montium species group, the
amount of saturated triacylglycerols was
smaller than the value expected on the
assumption that fatty acids are randomly
distributed in the triacylglycerols,
suggesting that non-random distribution of
unsaturated fatty acids among
triacylglycerols. This may facilitate the
lowering of the transition temperature of
triacylglycerols and hence may be related
to the ability of Drosophila to cope with
temperate climates.
COLD RESISTANCE OF CALCIUM TRANSPORT
ACTIVITY IN SARCOPLASMIC RETICULUM OF
SCALLOP STRIATED ADDUCTOR MUSCLE -----
--- CALCIUM-TRANSPORTING PROTEIN OR
THE MEMBRANE LIPIDS ?
D. Sato and J. Nakamura. Biol. Inst., Fac. of Sci.,
Tohoku Univ.
Different from the sarcoplasmic reticulum (SR)
of rabbit skeletal muscle, calcium-transporting
ATPase of scallop (Patinopecten yessoensis) SR has
been found to be able to operate at low temperature
(0-20 °C) and to be irreversibly inactivated at high
temperature (37 °C). Here , we. preliminarily studied
the role of lipid of scallop SR in the cold resistant
property. Temperature profile of the scallop ATPase
activity was compared with that of the rabbit ATPase
activity in the presence and absence of excess amount
of nonionic detergent (C]2E8). Thier temperature
profiles were not significantly affected by the
detergent. The cold resistance of scallop SR may be
based on the ATPase protein itself.
ATP ENHANCES CALCIUM-DEPENDENT
CALCIUM OCCLUSION IN CALCIUM-
TRANSPORTING ATPase OF SARCOPLASMIC
RETICULUM FROM RABBIT SKELETAL
MUSCLE
J. Nakamura, Biol. Inst., Fac. of Sci., Tohoku Univ.
In the previous meeting, we reported that
45Ca2+-40Ca2+ exchangeability of 45Ca bound to
the calcium transport sites of Ca2+-ATPase of the
sarcoplasmic reticulum is heterogeneous in the
absence of ATP: Half of the bound calcium is [Ca2+]-
dependent in a slowly exchageable (k <0.3 en).
"occluded" state in the Ca2+-ATPase, and the other
calcium is [Ca2+]-independent in a rapidly
exchageable (k=0.3 s~!), "unoccluded" state. Here,
the excangeability of the bound calcium was studied
in the presence of ATP at 0°C. By the addition of
ATP,the degree of the occlusion became higher
(k<0.003 s-1). The unoccluded calcium was,
however, not significantly affected. These results
suggest that ATP more highly occludes calcium,
already occluded in the absence of ATP.
Biochemistry 1143
MOLECULAR CLONING OF Na*,K*-ATPASE a-SUBUNIT
GENE IN THE SEA _ URCHIN, Hemicentrotus
pulcherrimus.
K. Yamazaki’, H.Kawashita’, C.Okamura’, kK.
Mitsunaga-Nakatsubo’, K.Yamada?, K.Akasaka?,
H.Shimada*® and I.Yasumasu’. ‘Dept. of Biol.,
Sch. of Educ., Waseda Univ., Tokyo, “Dept.
of Radiation Res., The Tokyo Metropol. Inst.
of Med. Sci., Tokyo, *Zool. Inst., Fac. of
Sci., Hiroshima Univ., Hiroshima.
We have isolated the cDNA of Na’*,K*-
ATPase a-subunit from embryos of the sea
urchin, Hemicentrotus pulcherrimus, and
determined its DNA sequence. Northern blot
analysis with the cDNA probe indicated that
this mRNA was expressed maximally at the
messenchyme blastula stage preceding the
increase of the activity. And its mRNA was
predominantly expressed in ectoderm cells.
To analyse the regulatory mechanism of
the expression of Na‘,K*’-ATPase in the stage
and cell-lineage, we have tried to isolate
and characterize the gene.
Since the gene encoding Na*,K*-ATPase a-
subunit seemes to be so long, two probes
were prepared, one of which encodes N-
terminal reagion and the other C-terminal
reagion. Ten positive signal clones were
isolated by screening 2.5 x 10° of sea
urchin genomic library with these probes.
And now we are analysing these clones.
MITOSIS -SPECIFIC PHOSPHORYLATION OF
DYNAMIN
S. KOMATSU!? , HHOSOYA!S,
T. SHIMIDZU4 ,M.INAGAKI° , M.|KEGAMI2,
K.YAZAKI! 1Tokyo Metro. Inst. Med. Sci.,
2Tokyo Agri. Univ ., 3 Hiroshima Univ is 4Re ser. Inst.
Polym. and Tex. Tokyo Metro. Inst. Geront .
Cytosol preparations form mitotically selected Hela
cells exhibited much higher kinase activities to
phosphorylate a 100kD microtubule cross-linking
protein, dynamin, than the preparations from non-
mitotic cells. Based on substrate specificity and lack
of sensitivities to characteristic activators or inhibitors,
this dynamin kinase activity appears to be distinct
from A-kinase, cGMP dependent protein kinase, C-
kinase or Ca2+-calmodulin dependent protein kinase
Il. Purified cdc2 kinase (p34°9°?-cyclin B complex)
phosphorylated dynamin. Microtubule-binding activity
of phosphoyrlated dynamin was reduced.
FURTHER CHARACTERIZATION OF DYNAMIN.
T. Shimizu", T. Kokubu', S. Ohashi’, Y.Y. Toyoshima?,
and T. Miki-Noumura?. ‘Res. Inst. Polym. Text.,
Tsukuba, Ibaraki, and 2Ochanomizu Univ., Dept. Biol.,
Bunkyo, Tokyo, Japan.
Dynamin (100k protein) isolated from rat brain high
speed supernatant exhibited GTPase activity higher than
other NTPase activity as reported (Shpetner & Vallee,
(1992) Nature 355, 733). The GTPase activity depended
upon pH, being high at an acidic pH (5.5 to 6) and low at
pH 7 or higher. It decreased upon increase in ionic
strength and was quite low at 0.2 M NaCl or higher. At
PH 6.7 in the absence of NaCl, the apparent Km for GTP
was 44M. Dynamin did not exhibit an initial phosphate
burst with 304M GTP or ATP. Its GTPase activity was
enhanced by microtubules (MTs) as reported by
Shpetner and Vallee.
Our previous dynamin preparation was shown to
contain up to 0.03% kinesin by immunoblotting with poly-
clonal kinesin antibodies. When kinesin contamination
was eliminated by the use of DEAE-Sephacel chromatog-
raphy, the resultant dynamin poorly translocated MTs.
Adaition of the original contaminating level of kinesin was
sufficient to restore MT movement. The previous obser-
vation of MT translocation by dynamin preparation, there-
fore, was likely to be due to kinesin. Thus, dynamin does
not interfere with the kinesin motility, but even supports
low density kinesin motility like casein or cytochrome c.
IMMUNO-ELECTORON MICROSCOPIC STUDIES ON
BETA- AND GAMMA- DYNEIN HEAVY CHAINS FROM
TETRAHYMENA CILIA. D 1
E.Masuyama', M,Ishida“, K.Nakamura’, and
Y.Shigenaka?. 1 Dep, gf Living Sci.,Univ.
of Hiroshima Women's, Fac. Integr. Arts
& Sci., Univ. of Hiroshima, Hiroshima,
When Tetrahymena Ciliary 22S dynein was
digested wi ermolysin, two protease-
resistant_fragments which were designated
TH-1 and TH-2 were obtained.The fragments
were characterized by high ATPase acti-
vities and consisted of two principal
polypeptids (TH-1: 173K and 80K, TH-2:
173K and 120K). Polyclonal antibodies
against these dynein fragments were
prepared and used to investigate the
Origin of the fragments and the
Structural relationships between the
polypeptides of the fragments. By immuno-
Dlotting analysis, anti-TH-1 and anti-TH-
2 crossreacted strongly with the gamma-
and beta-heavy chain of 22S dynein,
respectively. The two antibodies
recognized only the 173K polypeptide of
each fragment. We also used these
purified antibodies to further
investigate the location of beta- and
gamma-dynein heavy chains within the
axoneme. In immuno-electron micrographs
we observed that these antibodies
labelled with Protein A-gold particles
attached to outer arm region. Furthermore
the two gold particles located in
Slightly differing positions of the outer
arm.
1144 Biochemistry
MOLECULAR CLONING OF CYTOPLASMIC
DYNEIN cDNA
K. Ogawal and T. Shimizu2. 1Natl. Inst. for
Basic Biol., Okazaki and ?Res. Inst. of Polym.
Text., Tsukuba.
Bovine brain cDNA library was screened by
affinity-purified anti-MAPI1C (cytoplasmic form
of dynein) antibody. One clone among 14 positive
candidates gave a signal corresponding to 15 kb
poly(A)+RNA in Norther blot. The signal was
detected in human brain, heart, muscle and liver
poly(A)+RNA. Sequencing showed that this clone
(AJ073) encodes for the carboxy terminus of a
protein. Homology search by MAILBLAST
program picked up the carboxy terminal sequence
of dynein B heavy chain of sea urchin. Therefore,
we consider that this clone corresponds to the 3'-
terminal part of cytoplasmic dynein heavy chain
cDNA.
CENTRAL-PAIR MICROTUBULES OF CHLAMYDOMONAS
FLAGELLAR AXONEMES HAVE DYNEIN ATPase OR
NOT.
N.Ishii and T.Miki-Noumura. Department of
Biology, Ochanomizu Univ., Ohtsuka, Tokyo.
Little is known about the function of
central-pair microtubules(central-pair MTs)
in flagellar movement. We isolated central-
pair MTs from Chlamydomonas flagellar
axonemes (Hosokawa and Miki-Noumura,1987)
and examined the polypeptide composition,
attempting to make clear existence of
dynein ATPase in them.
We examined here the polypeptide
composition in 7.5% SDS-polyacrylamide gel
electropholesis. The central-pair MTs
consisted of about 25 polypeptides, of
which major one was tubulin. Using 4% and
3-5% gradient gel, we examined the bands in
high=-molecular-weight region. Any bands
corresponded to dynein was not found in
the polypeptides of central-pair MTs.
Next, we carried out immunological
analysis of dynein bands, using antibodies
against Tetrahymena and Chlamydomonas
dynein heavy chain. The band in the high-
molecular-weight region of the central-pair
MTs did not react with these antibodies.
Measurement of ATPase activity also
showed no activity in central-pair MTs.
The results, analysis of polypeptide
bands, Western blot, and ATPase
measurement, suggest that the central-pair
MTs may not have dynein ATPase.
KINESIN-DEPENDENT MOTILITY AND MICRO-
TUBULE STRUCTURES.
S. Kamimura! and E. Mandelkow~. Dep.
Biol., Coll. Arts & Sci., Univ. Tokyo,
Tokyo and *Max-Planck-Unit, Struct. Mol.
Biol., c/o DESY, Hamburg, Germany
Microtubules are build of tubulin
subunits assembled into hollow cylinders
which consists of parallel protofila-
ments. Thus, kinesin interacting with a
microtubule could do so either with one
or several tubulin units. This makes it
difficult to determine the structural
requirements for the interaction
One way to approach the problem is
to alter the surface lattice. This can be
done in several ways. Protofilaments can
be exposed on their inside (C-tubules),
they can be made antiparallel (zinc
sheets), or they can be rolled up (duplex
tubules). We have exploited this polymor-
phism to study how kinesin attached to a
glass surface interacts and moves the
various tubulin assemblies.
About 40% of C-tubules glided
similarly to microtubules. Zinc Sheets
showed motility but the motion was dis-—
continuous. Duplex tubules did not move.
This indicates (a) that a full cylindri-
cal closure of microtubule is not neces-
sary for movement, and (b) that the
inside surface of microtubules does not
support motility. The data can be ex-
plained by assuming that the outside of
one protofilament represents the minimal
track for kinesin.
(ref. ) Kamimura, S. & E. Mandelkow
(1992) J. Cell Biol., 118:865-875.
STUDIES ON TUBULIN ISOFORMS INVOLVED IN
AXONEMAL FUNCT ION OF TETRAHYMENA CILIA.
K. Nakamura E. Masuyama’, S. Wada“, and
M. Okuno*. ‘Dept. of living sciences,
iroshima Women's Univ., Hiroshima,
Dept. of Biol., Col. of Arts and Sci.,
Tokyo Univ. Tokyo
SS ea TOTS RONEM ET DT OTE TI
consists of two subunits (alpha and
beta-tubulins). Each tubulin subunit is a
heterogeneous protein composed of a mix-
ture of isotubulins. In order to investi-
gate PHO CEHON La SSten SHS a SSDs
lins on ciliary movements of [etra eM,
it is essential to identify ese
isoforms and to study their roles in
axonemal movement. We have presented two
novel methods which are useful for these
analysis. One is the native gel
electrophoresis with agarose and acryl-
amide gel. Using this method and
immunoblotting techniques with anti-
tubulin antibodies, we have shown that
tubulin molecules can interact with
several proteins including dyneins. The
other method is two-dimensional electro-
phoresis designed for the separation of
tubulin isoforms. This method uses 1so-
electric phocusing at the second dimens-
ion. At least 10 tubulin isoforms from
Tetrahymena axonemes can be detected by
this new two-dimensional electrophoresis.
Using these electrophoretic systems, we
have suggested that a beta-isotubulin,
tentatively called as Ib, can interact
with axonemal proteins including dyneins.
Biochemistry 1145
PURIFICATION OF PROTFASOMES FROM SALMONID
FISH SPERM AND THEIR LOCALIZATION ALONG
SPERM FLAGELLA.
K. Inaba, M. Morisawa and Y. Akazome.
Misaki Marine Biological Station, Faculty
of Science, University of Tokyo, Miura,
Kanagawa.
Motility of demembranated sperm in
salmonid fish is inhibited by chymotrypsin
inhibitors in an ATP-dependent manner. We
purified chymotrypsin-like proteases from
chum salmon sperm, with the molecular
masses of 950 and 650 kD. Some enzymatic
properties and molecular shape of the 650
kD protease showed that this protease was a
multicatalytic proteinase (proteasome) as
is well known to participate in ATP-
dependent degradation of ubiquitinated
proteins. We prepared polyclonal antibody
against purified 650 kD proteasome. This
antibody recognized mainly the 29 and 28 kD
subunits of proteasome. Using immuno-
fluorescent microscopy, we examined the
subcellular localization of proteasomes in
sperm. The result showed that proteasomes
are located predominantly in sperm flagella.
Interestingly, the anti-proteasome antibody
did not stain overall portion of sperm tail
but showed patches or somewhat periodical
staining patterns along sperm flagellum.
These results suggest that activation of
proteasomes at regular intervals on sperm
Elagellum causes ATP-dependent conversion
of microtubule sliding to flagellar bending,
resulting in the regulation of sperm
motility.
MITOSIS-SPECIFIC PHOSPHORYLATION OF
MICROTUBULE ASSOCIATAED PROTEINS
F. TAKAHASHI!» , H. HOSOYA!:3 , K. KOBAYASHI‘ ,
M. INAGAKI? , T. ARAF. , K. YAZAKI!
'Tokyo Metro. Inst. Med. Sci 42 Tokyo Sci. Univ .,
3 Hiroshima Univ a 4 Jikei Univ 3 “Tokyo Metro.
Inst. Geront .
Mitotic Hela cell extracts exhibited much higher
kinase activities to phosphorylate brain microtubule
associated proteins (MAP2 and MAPtc) than the
extracts from non-mitotic cells. This MAP2/MAP1c
kinase activity appears to be distinct from A-kinase,
cGMP-dependent protein kinase, C-kinase or Ca¢t-
calmodulin dependent protein kinase Il. Purified
cdc? kinase (p34°9°?_cyclin B complex)
phosphorylated MAP2 and MAP‘1c.
PROTEIN PHOSPHORYLATION IN POLYMORPHO-
NUCLEAR LEUCOCYTES RELATED TO THE SIGNAL
TRANSDUCTION OF CHEMOATTRACTANT
STIMULATION
M.Shibata!, T.Ohoka!, Y.Yamakawa2, S.Mizuno2 and
K.Suzuki2. !Dept.Biol., Fac.of Sci., Tokyo Metropol. Univ.
and 2Nat. Inst. of Health.
Human polumorphonuclear leucocytes (PMN) are known to
be activated by various kind of chemoattractants, LUCT/IL-8,
leucotriene B4 or FMLP, lead to migration, phagocytosis or O27
production. To elucidate the molecular mechanism of signal
transduction, phosphorylated proteins are analyzed by two-
dimensional electrophoresis and autoradiography, after the 32P-
labelling of intact PMN and stimulation with FMLP . Protein
subunits having molecular weight of 82, 66, 64, 58, 55 and 50
kDa were able to be detected, the marked phosphorylation was
observed with 64 kDa proteins. One of the 64 kDa proteins are
revealed to be phosphoglucomutase, this phosphorylation was
stimulated in presence of a micromolar level of glucose, by a
mechanism including hexokinase and substitution of 32P-
phosphate from glucose-6-phosphate to active site seine. The
other phosphorylatable 64kD protein (p64) was detected after
FMLP stimulation, having a isoelectric point (pl=5.3) different
from phosphoglucomutase. The pl shifted after phosphorylation
from 5.3 to more acidic side forming pp64. The FMLP-
stimulated phosphorylation was time-dependent and saturated
within 5 min., the maximum stimulation was achieved with 10
nM FMLP. Phosphoamino acid analysis of the pp64 revealed
the phosphorylation of the serine residue. Staurosporine (100
nM) and W-7 (100 mM) significantly inhibited the
phosphorylation, H-7 slightly inhibited, H-8 and herbimycin did
not affect. These data suggested that protein kinase C and
calmodulin like protein(s) are concerned. From the purification
studies of p64 and by amino acid analysis, p64 was identified as
]-plastin, one of the leucocyte specific proteins. Phosphorylation
of this may play a role in cytoskeletal reorganization of PMN.
INDUCTION OF AMOEBOID MOVEMENTS IN THE SEA
URCHIN EGG BY PROTEIN PHOSPHORYLATION.
-Mabuchi-, A.Morimatsu and H.Tosuji~.
Dept. of Biol., Coll. of Arts and Sci.,
Winsk\7o Ore YON WO, Dept. Biophys. and
Biochenm., ac. of Sci.,Univ. of Tokyo,
Tokyo and ~Dept. of Biol., Fac. of Sci.,
Kagoshima Univ., Kagoshima.
Calyculin A, an inhibitor of protein
phosphatases type 1 and type 2A, induces
cleavage-like changes in the unfertilized
sea urchin egg (Tosuji et al., Proc. Nat.
Acad. Sci. USA, in press). It induces
formation of protrusion or amoeboid
movements in the fertilized eggs. We
recently found that calyculin A induces
the amoeboid movement also in the
unfertilized eggs at appropriate
concentrations. These eggs usually did
not cleave, but cleavaed by the addition
of IBMX or TPA. TPA alone could induce
the movements in the fertilized eggs.
Tautomycin, which has been known to be
another inhibitor of protein phosphatases
type 1 and type 2A (Hori, M. et al., FEBS
Lett., 285: 145-148, 1991) also induced
the movements in the fertilized sea urchin
eggs. These results strongly suggest that
protein phosphorylation is involved in the
cytoskeletal organization in the sea
urchin egg.
1146 Biochemistry
EFFECTS OF WORTMANNIN, AN INHIBITOR OF MYOSIN
LIGHT CHAIN KINASE, ON THE CELL DIVISION OF
CULTURED CELLS OR SEA URCHIN EGGS
H.HOSOYA |: 2, ETAKAHASHI 2° ©, M.INAGAKI 3,
Y.MATS UDA 4, Y.NONOMURA ®, TARAI ©,
E.NAKAMURA 2: 7 H.SAKAI ”, K.YAZAKI 2
THiroshima Univ., 2 Tokyo Metro. Inst. Med.Sci.,
STokyo Metro. Inst. Geront., 4kyowa Hakko Inc.,
Suniv. Tokyo, °Tokyo Sci. Univ.,/Japan Women's Univ.
We investigated roles of myosin light chain kinase
(MLCK) on cell division by using a specific inhibitor
of MLCK, wortmannin. Wortmannin inhibited
cytokinesis with a half maximal inhibition at 1-2 pM.
The nuclear division was accomplished normally at
10u.M where the cytokinesis was completely blocked.
Fluorescent staining of actin filaments with rhodamine-
labeled phalloidin revealed that the contractile ring
was not formed in the cleavage-inhibited eggs.
Wortmannin did not inhibit cdc2 kinase activity.
ELECTRON MICROSCOPY OF 1200 kDa
FRAGMENT OF a -CONNECTIN FROM RABBIT
SKELETAL MUSCLE
J. Suzuki, S. Kimura and K. Maruyama.
Dept. Biol., Fac. Sci., Chiba Univ., Chiba.
a -Connectin, an elastic protein, of striated muscle
is hydrolyzed into £ -connectin and 1200 kDa peptide
in situ, when exercised muscle is for about 15 h kept at
AT.
In the present work, rotaty shadowed images of the
isolated 1200 kDa peptide were examined under an
electron microscope. There were a main peak at 350
nm and a smaller peak at 170 nm in the length
distribution. The width of the former was 3 nm and
that of the latter was 4.5 nm suggesting that the latter
was folded form of the former. It is concluded that the
1200 kDa peptide was a filament 350 nm long and 3 nm
wide.
Under the same conditions, £-connectin was
observed as a filament 750 nm long. The molecular
mass of £-connectin has been estimated to be
approximately 2700 kDa. The ratio of molecular mass
(1200 : 2700 = 1 : 2.3) was similar to that of molecular
length (350 : 750 = 1 : 2.1).
EPTOPE MAP OF ANTIBODIES TO CONNECTIN
CHICKEN STRIATED MUSCLE.
Y. Kawamura, Y. Ohtani, S. Kimura and K. Maruyama. Dept.
Biol., Fac. Sci., Chiba Univ., Chiba.
Connectin molecule,sMW,~3000,000,links the myosin
filament to the Z line a sarcomere of vertebrate striated
muscle.Immunoelectron microscopy reveals striation in a
sarcomere due to the binding of an antibody to a specific epitope
in connectin, because connectin filaments run parallel with the
mainaxis of a sarcomere.
We have examined the specific epitope sites of 11 kinds of
monoclonal and 1 kind of polyclonal antibodies to connectin in
chicken breast muscle. The results obtained were compared with
those reported by K.Weber's and J.Trinick's groups. We have
also compared the epitope locations in chicken breast muscle
with those in chicken leg muscle. There were the cases where the
epitope region was different between the two types of chicken
skeletal muscle .
SECONDARY STRUCTURE OF MUSCLE ELASTIC
PROTEIN CONNECTIN AS REVEALED BY RAMAN
SPECTROSCOPY.
Y. Nakauchi', K. Maruyama’, S. Toyama?, and I. Harada’. ‘Dept.
of Biol., Fac. of Sci., Chiba Univ., Chiba, *Fac. of Pharmac.,
Tohoku Univ., Sendai.
It has been reported that muscle elastic protein connectin
consists of 60% B-sheet and 30% f-turn based on circular
dichroism measurements (Maruyama er a/., 1986). The present
work using Raman spectroscopy extends the previous work.
Ultraviolet resonance Raman spectra of B-connectin in solution
indicated the presence of B-sheets and hydrogen-bonded
irregular structures. Infrared spectra showed an abundance of B-
sheets in B-connectin fiber, some of which were aligned with
their main chain axes parallel to the fiber axes. Thus, the B-spiral
structure proposed for elastin is improbable in connectin.
We have also obtained infrared Raman spectra of a 1200 kDa
peptide fiber. The 1200 kDa peptide which constitute elastic
portion of connectin in situ was also rich in B-sheet structure.
We propose a model of connectin structure based on the
repetitive amino acid sequence of connectin (Labeit er a/., 1990,
1992) and also our finding.
Biochemistry 1147
CONNECTIN FILAMENTS OF GTANT SARCOMERES OF
CRAYFISH CLAW MUSCLE
T. Manabe, H. Higuchi?, Y. Kawamura’, S. Kimura’, and K.
Maruyama’. 'Dept. of Biol., Fac. Sci., Chiba Univ., Chiba, "Dept.
of Physiol., Jikei Univ. Sch. Med., Tokyo
In giant sarcomeres (10 jum at rest of crayfish claw muscle),
there is 3000 kDa protein in addition to 1200 kDa projectin. The
rotary shadowed image of the isolated 3000 kDa protein was
very thin filament, maximumly 0.9 jum long.
When skinned opener fibers were stretched up to 14 um
sarcomere length, resting tension developed, but on mild
treatment with trypsin resulted in the decrease in tension
development accompanied by splittin g of the 3000 kDa protein.
Immunofluorescence microscopy using anti-3000 kDa protein
antibodies revealed that the protein linked the Z line to the edge
of the A band and it was movable devending on the sarcomere
length.
From the present study, it is concluded that the 3000 kDa
protein in the giant sarcomeres of crayfish craw muscle
corresponds to connectin, elastic protein of vertebrate skeletal
muscle.
LOCALIZATION OF TWO MYOSIN ISOFORMS IN
PERITONEAL NEUTROPHILS FROM GUINEA PIG.
H.Takano-Ohmuro, M.Endo and K.Kohama*
Dept. of Pharmacol.Fac. of Med., Univ.of
Tokyo, Tokyo, and “Dept. of Pharmacol. Fac. of
Med., Gunma Univ. Maebashi
We previously reported that peritoneal
neutrophils of guinea pig has two isoforms
of myosin Il differed in the heavy chain
(HC) (Zool. Sci., 1991)
To localize the isoforms, we subjected
the cell lysate of the neutrophils to the
centrifugation at 120,000 g for 70 min. The
supernatant was used as cytosolic fraction
(Cyt). The precipitate (ppt) was suspended
in an isotonic solution and the suspension
was centrifuged at 10,000 g for 30 min. The
ppt after 10,000 g centrifugation was used
as 10K fraction (10K) and the suspension was
further subjected to the centrifugation at
120,000 g for 70 min. The ppt was used as
120K fraction(120K). When Cyt, 10K and 120K
were subjected to native pyrophosphate gel
electrophoresis, they showed only one band.
The mobility of myosin in cytosol did not
coincide with that in 10K. From peptide
Mapping and immunoreactivity, the difference
could be explained by the difference in HC
of both myosin. Myosin of Cyt comigrated
with that of 120K. Our similar analyses
showed that myosin of Cyt was identical
in HC and light chains with myosin of 120K.
The reason to explain the difference in
the localization between Cyt and 120K
remained to be examined.
MYOSIN FROM THE SEA SPONGE, Halicondria
okadai
N. Kanzawa and K. Maruyama. Dept. Biol., Fac. Sci.,
Chiba Univ., Chiba.
We have attempted to purify myosin from the sea
sponge, Halicondria okadai. There has been no report
on sponge myosin.
Sea sponge myosin consisted of 220 kDa heavy chain
and two species of light chains, 18 and 21 kDa. Two
headed structure was observed under electron
microscope. The K-EDTA activated ATPase activity
was as high as 0.5 imole/mg/min, but the Mg?*-ATPase
activity was low and only slightly enhanced by rabbit
F-actin.
Solubility of sea sponge myosin was lower than
rabbit skeletal myosin: the former was only soluble by
40% at 0.3 M KCl as compared to the latter (by 80%).
Sea sponge myosin formed thick filaments, 0.5-1 [1m
long, at 0.3 M KCl, where rabbit skeletal myosin
formed much smaller oligomers.
ON THE TISSUE-SPECIFIC DISTRIBUTION OF
TROPOMYOSIN ISOFORMS IN CRUSTACEAN MUSCLES
T.Ishimoda-Takagi, S.Nakano, K.Hino and M.
Itoh. Dept. of Biol., Tokyo Gakugei Univ.,
Tokyo.
We have previously shown that several
species of tropomyosin (TM) isoforms were
included in the spiny lobster and American
lobster. Distribution of the TM isoforms
was tissue specific, and tissue-specifici-
ty of the TM isoforms correlated consider-
ably with the types of muscle fiber. In
order to investigate generality of the
tissue-specificity of TM isoforms in crus-
tacean decapod, we examined tissue speci-
ficity of TM isoforms in the prawn,
Panaeus japonicus, and the crab, Erimacrus
isenbeckii. Four TM isoforms were present
in the prawn muscles. Distribution pat-
tern of TM isoforms was similar to that
of spiny lobster. The TM component a was
a main component of the cephalo-thoracic
and abdominal muscles. The component b
waS mainly observed in the leg muscle.
The component c was mostly involved in the
dorsal muscles. Heart muscle contained
a heart-specific TM isoform. The TM com-
ponent corresponding to b was also observ-
ed in the crab leg muscle. Furthermore,
crab heart muscle contained heart-specific
TM isoforms. However, the TM isoforms
corresponding to the components a and c,
which were involved in the muscles to move
abdomen, could not be identified in the
crab of which abdomen is greatly reduced.
1148 Biochemistry
DNASE I-BINDING PROPERTY OF CHIMERIC
ACTINS CONSISTING OF TETRAHYMENA ACTIN
AND DICTYOSTELIUM ACTIN
M. Hirono!, K. Sutoh?2, Y. Watanabe? and Tadao
Ohno!. !RIKEN Cell Bank, Institute of Physical and
Chemical Research, Tsukuba, 2Department of Pure
and Applied Sciences, College of Arts and Sciences,
University of Tokyo, Tokyo, “Institute of Biological
Sciences, University of Tsukuba, Tsukuba.
Tetrahymena actin has a unique primary
structure and some unusual properties such as
lacking a DNase I-binding activity. To know which
sequence of Tetrahymena actin corresponds to those
unusual properties, we constructed two chimeric
actin genes from Tetrahymena actin gene and Dicty-
ostelium actin gene, and those genes were expressed
in Dictyostelium cells. One chimeric actin (designat-
ed as Tet84Dic) was contracted from N-terminal
portion (1-83 residues) of Tetrahymena actin and C-
terminal portion of Dictyostelium actin (84-375
residues). The other chimeric actin (designated as
Dic84Tet) was constracted from N-terminal portion
of Dictyostelium actin C-terminal portion of Tet-
rahymena actin connecting at the same site as
Tet84Dic. Using DNase I-affinity column, we re-
vealed that one of the chimeric actin gene product,
Tet84Dic, was not retained in the column whereas
intrinsic actin was retained. On the other hand, we
also found that Dic84Tet was retained in the DNase
I column. In conjunction with our previous data that
Tetrahymena actin does not interact with DNase I,
we suggest that the binding site of DNase lina
ubiquitous actin is located in N-terminal region
(residues 1-83).
STRUCTURAL CHANGES OF ACTIN FILAMENTS
INDUCED BY GONIODOMIN A
S. Watanabe’, K. Maruyama!, K. Furukawa’, and Y. Ohizumi?.
Dept. Biol., Fac. Sci., Chiba Univ., Chiba, 7Fac. Pharm. Sci.,
Tohoku Univ., Sendai.
Goniodomin A purified from Goniodoma pseudogoniaulax
increases the actin—activated Mg?*-ATPase activity of myosin
at 107M and inhibits it at 10° M [ Ohizumi,Y., Folia
pharmacol. japon. 100, 259-269 (1992)].
In the present study, the effect of goniodomin A on actin
was investigated. Goniodomin A, 10° M, hardly affected
fluorescence intensity of pyrenyl actin (Ex 365nm; Em
407nm), but at 3 x 10° M the intensity was remarkably
dropped ( to 8% of the control). In the presence of 3 x 10°M
goniodomin A, actin filaments were associated with each other
to form a gel. Therefore, the aggregates were completely
sedimented by centrifugation for 15 min at 12,000 x g.
The present results sugggest that goniodomin A
stoichiometrically bound to actin monomers in the actin
filament and conformational changes of actin induced by
goniodomin A may affect its interaction with myosin.
A HIGH MOLECULAR WEIGHT PROTEIN FROM
CHICKEN GIZZARD SMOOTH MUSCLE.
A. Terasaki and K. Ohashi. Dept. of Biol.,
Fac. of ‘Sci., Chiba Univ., Chiba.
Myofibrils of chicken gizzard smooth
muscle were extracted with a high salt
solution. Successively the residue was
extracted with a low salt alkaline solu-
tion (pH 9.0). The high molecular weight
protein, whose molecular weight was more
than 400,000, was not extracted from the
residue at 4 °C but 37 °C. The extract
was fractionated by ammonium sulfate. The
precipitate of 20% saturation was suspend-
ed in a solution containing 20 mM NaCl (pH
8.0) and dialyzed against the same buffer.
A small part of the high molecular weight
protein could be redissolved. The solu-
tion was applied on a hydroxylapatite
column. The high molecular weight pro-
tein was eluted at the phosphate concen-
tration of 0.15 M. The eluant was chro-
matographed on a DEAE-Cellulofine column.
The high molecular weight protein was
eluted at the NaCl concentration of 0.25 M
with a small amount of actin. This frac-
tion was rechromatographed on a DEAE-
Cellulofine column in the presence of 4 M
urea to remove actin, The yield was 0.05
mg from 100 g of smooth muscle, The
antibody against the high molecular weight
protein stained the adhesion plaques of a
cultured cell. Immunoblot analysis showed
that the high molecular weight protein
were present in smooth (gizzard), skeletal
(breast), and cardiac muscles.
33KD PEPTIDES FROM CHICKEN GIZZARD SMOOTH
MUSCLE.
H.Nakagawa, M.Ishihara and K.Ohashi.
Dept. of Biol., Fac. of Sci., Chiba Univ.,
Chiba.
We prepared a pair of peptides, which
were associated with actin and desmin,
from chicken gizzard smooth muscle. Their
apparent molecular weight on SDS-PAGE was
approximately 33,000. Myofibrils of chick-
en gizzard smooth muscle were extracted
with Hasselbach-Schenider’s solution.
Solid ammonium sulfate was added into the
extract to 70% saturation. The supernatant
was dialyzed against 0.1 M KCl. A small
amount of precipitation was coilected by
centrifugation and solubilized in 0.6 M
KCl. The main components of this solution
were’ the 33kD peptides. The yield of the
33kKD peptides was 0O.5mg from 100g of
smooth muscle. The two peptide bands on
SDS-PAGE were stained purple with CBB-R250
and easily distinguishable from the other
blue peptide bands. The electron micro-
scopic observation showed that the 33kD
peptides bundled F-actin and formed dense
aggregates of desmin filaments. The 33kD
peptides were coprecipitated with F-actin
and desmin filaments by centrifugation at
15,000 rpm. When G-actin and desmin proto-
filaments were assembled in a _ solution
containing the 33kD peptides at the same
time, F-actin bundles decorated with many
dense aggregates of desmin filaments were
observed under an electron microscope.
Biochemistry 1149
LOCALIZATION OF A 52KD PEPTIDE FROM CHICK-
EN GIZZARD SMOOTH MUSCLE IN VARIOUS TYPES
OF CELLS.
H. Nakagawa, M. Nishimura, and kK. Ohashi.
Dept. of Biol., Fac. of Sci., Chiba Univ.,
Chiba.
A 52kD peptide, purified from chicken
gizzard smooth muscle, had a weak interac—
tion with actin fragments and localized at
the leading edges of a moving cultured
smooth muscle cell. Immunoblot analysis
showed that the 52kD peptide was present
in various types of tissues and cells of
chicken, i.e. skeletal (breast muscle),
cardiac, and smooth (gizzard) muscles, and
nonmuscle tissues or cells (brain, small
intestinal epithelial cells, and lympho-
cytes). By immunofluorescence method, the
antibody against 52kD peptide was strongly
stained the junctional complex of an
isolated small intestinal epithelial cell
and also seemed to stain the costameres of
a skeletal muscle fiber. In these cells,
immunofluorescence of anti-52kD peptide
antibody was observed at the vicinity of
the plasma membranes. In cultured smooth
muscle cells, the distribution of fluores-
cence partially coincided with the stress
fibers and cell edges. In addition, the
distribution had a granular and a vesicu-
lar component which are seen in both the
perinuclear and peripheral regions of the
cell. In cultured cardiac muscle cells,
the vesicular components were rich in the
contact regions of neighboring cells.
CLONING AND SEQUENCING OF cDNA ENCODING
CHICKEN PROFILIN. R. Mochizuki, N. Minami,
H. Abe and T. Obinata. Dept of Biol., Fac.
of Sci., Chiba Univ., Chiba.
In the previous studies, we isolated
and characterized an actin-binding protein
of 16 kDa (profilin) from embryonic
chicken skeletal muscle, which sequesters
monomeric actin in the cytoplasm, and have
demonstrated that this protein regulates
differentially the assembly of
cytoskeletal (8,y ) and sarcomeric (a )
actins during myofibrillogenesis. In the
present study, we cloned cDNA encoding the
profilin from the cDNA library prepared
with poly(A ) RNA from embryonic chicken
skeletal muscle by using specific
antibodies as probes, and determined the
sequence. The deduced amino acid sequence
was compared with previously described
non-muscle profilin sequences. The
chicken sequence exhibited about 77 %
identity with those of profilins from
human and bovine non-muscle tissues. The
similarity of the chicken profilin with
those from echinoderm, amoeba, and Physulm
was very low; 19 Z% identity with
Clypeaster profilin and 17 % identity with
Physulm profilin. Nevertheless, fifteen
residues conserved beyond the species were
observed. Northern RNA blot analysis
showed that the expression of profilin is
down-regulated in parallel with that of
cytoskeletal (8 , Y ) actins during deve-
lopment of chicken skeletal muscle.
PURIFICATION OF DOMAIN-SPECIFIC ANTIBODIES
USING AFFINITY CHROMATOGRAPHY AND SDS-
PAGE.
Y. Yoshihara and M. Kuroda. Dept. of Biol., Fac.of Sci.,
Shimane Univ., Matsue.
By a combination of SDS-PAGE and affinity chromato:
graphy, we have developed a new method to isolate domain-
specific antibodies from polyclonal antiserum. Chymotryptic-
digests of @—actinin was separated by SDS-PAGE. The 32K-
head and 55K-rod bands were cut out from CBB-stained gels
respectively. Gel pieces containing 32K or 5SK domain were
placed on CNBr-activated Sepharose packed in a short glass
tube. The 32K or 55K polypeptide was electro-phoretically
eluted from gel pieces onto the CNBr-activated Sepharose.
Then the Sepharose gel was used as an immuno-absorbant.
Undigested 104K subunit of a-actinin was also treated in the
similar way. Goat anti-chicken skeletal @-actinin antiserum
was applied to these three affinity columns. The anti-55K IgG
that had eluted from the 55K-rod conjugated affinity column
reacted with all the rod-containing subfragments in chy-
motryptic digests but did not cross-reacted with 32K-head
fragments at all. Using 1D-peptide map of the 55K-rod
domain, we assayed the epitope of the anti 55K IgG between
Pro360-Arg502 of a@—actinin. Meanwhile, anti-32K IgG
reacted slightly with some of the rod containing subfragments
beside 32K-head. We assume that the anti-32K IgG
recognized head-rod junction of @-actinin.
ACTIN CYTOSKELETON IN CULTURED MYOGENIC
CELLS TRASNFECTED WITH COFILIN cDNA.
S. Ono, H. Abe and T. Obinata. Dept. of
Biol., Fac. of Sci., Chiba Univ., Chiba.
Cofilin is an actin-binding protein
which modulates actin assembly in both
muscle and non-muscle cells. To better
understand the role of cofilin in myogenic
cells, cofilin cDNA cloned into an
expression vector was transfected into
mouse C2 myogenic cells. We examined the
effects of cofilin over-expression on the
cellular morphology and actin assembly in
C2 cells. Under standard culture
conditions, alteration in actin filaments
was not clear as examined by phalloidin-
staining, although increased cofilin
expression was confirmed by staining the
cells with anti-cofilin antibody (MAB-22),
but the cellular shape was slightly
changed to become flat. When the
transfected cells were incubated in the
medium containing 10 % dimethylsulfoxide
(DMSO), actin filaments were remarkably
decreased and cofilin/actin rods were
formed in the nuclei. More drastic results
were obtained by heat shock, 1 hr at 43 C;
actin filaments were almost completely
disrupted and huge actin/cofilin rods were
generated in the cytoplasm and the nuclear
rods as well. A calmodulin inhibitor, W/,
also caused disassembly of actin
filaments. These results suggest that
cofilin become active in the cells treated
by heat shock or DMSO.
1150 Biochemistry
PHOSPHORYLATION AND DEPHOSPHORYLATION OF
COFILIN IN CULTURED FIBROBLASTIC CELLS.
H. Abe, K. Okada, and T. Obinata. Dept. of
Biol., Fac. of Sci., Chiba Univ., Chiba.
Cofilin, a 20 kDa actin-regulatory
protein, seems to be profoundly involved
in the assembly and disassembly of actin
filaments in cells. When cultured cells
are exposed to heat or incubated in a
medium containing 10 % dimethylsulfoxide
(DMSO), intranuclear actin-cofilin rods
are formed. Recently, it was demonstrated
that dephosphorylation of cofilin
accompanies its nuclear localization in
the cells treated with heat or DMSO. In
this study, we examined the
phosphorylation state of cofilin and its
cellular localization in Balb/e 3T3 and
rat 3Y1 cells in a synchronous’ culture.
When cells were arrested at GO phase by
serum starvation, the amount of
phosphorylated cofilin was significantly
decreased, although about 50 % of cofilin
was phosphorylated in an asynchronous
culture. Most of cofilin was present in
the cytoplasm of GO-arrested cells, as
examined by immunofluorescence microscopy.
The intranuclear actin-cofilin rods were
induced within 15 min by treating the
cells at the GO state with 10 % DMSO, but
dephosphorylation of cofilin was not
detected in this case. Therefore, we
conclude that dephosphorylation of cofilin
and its nuclear localization are
independent phenomena.
A STRUCTURAL STUDY OF A COFILIN ACTIVE
SITE ANALOG PEPTIDE.
Susumu Kotanil, Yutaka Ito’, Yutaka Muto2,
Eisuke Nishida@, and Shigeyuki Yokoyama“.
IDept. of Biochem. Eng. Sci., Kyushu Inst.
Tech., lizuka, and Dept. of Biophys.
Biochem., Fac. Sci., Univ. of Tokyo,
Tokyo.
Cofilin is a low molecular mass actin-—
regulating protein. A cofilin active site
analog dodecapeptide was analyzed by a
series of two-dimensional 1H-NMR methods
to determine the conformation. The pep-
tide proton resonances were unequivocally
assigned. The peptide alone in solution
contained no detectable secondary struc-
ture. However it takes a specific confor-
mation when mixed with actin, as revealed
by the transferred nuclear Overhauser
effect (TRNOE) measurements. The intra-
residue TRNOE’s were observed for all the
12 amino acid residues, suggesting that
the dodecapeptide interacts with actin and
PIPo along its entire length. The confor-
mation of the cofilin active site was
calculated using inter-residue and intra-
residue NOE data. The dodecapeptide N-
terminal five residues are folded into a
turn structure, while the C-termical port-
ion is extended.
DESMIN NET SURROUNDS MYOFIBRILS: DETECTION
BY IMMUNOELECTRON MICROSCOPY.
M. Kuroda !, Y Jinguhji 2 and K.Fujiwara 2. 1 Dept. of Biol.
Fac. of Sci., Shimane Uniy., Matsue, 2 Dept.of Strut Anal.,
Natl. Card. Cent. Res. Inst., Osaka.
In the previous meeting, we showed that the distribution of
desmin in chicken skeletal muscle was not restricted to Z-disks.
We showed following two fluorescence patterns of desmin
Staining that were significant especially when myofibrils were
bundled side by side; (i) longitudinal staining between adjacent
myofibrils in the bundle and (ii) diffuse staining on an entire
myofibril or over a bundle of myofibrils. These fluorescence
patterns suggested that desmin was not confined around Z-
disks but surrounded the surface of myofibrils. This time we
confirmed these observations by immunoelectron microscopy.
As desmin was expected to distribute on the surface of myofib-
rils, semithin unstained sections were examined using protein
A colloidal gold probes. In cross-sectional images of muscle,
Au-particles were detected around the periphery of each
myofibril and in the inter-myofibrillar space. No decoration
was Seen inside the cross-sectioned myofibrils. Meanwhile, in
longitudinally sectioned images, continuous arrays of Au-
particles distributed on myofibril besides Z-disk regions. Some
decorated filaments obliquely crossed over myofibrils on an
A- or I-band region. Branching of desmin filaments was a
common figure. Consequently, desmin filaments intercon-
nected each other to form networks around myofibrils.
PURIFICATION METHOD FOR Fg, FROM SOLUBLE
VITELLIN BINDING PROTEIN FROM LOCUSTA
MIGRATORIA
K. Yamasaki,
Dept. Biol. Tokyo Metropol. Univ. Tokyo.
Two types of vitellin binding activity have
been reported for the ovary of Locusta
migratoria. One of vitellin binding protein
(VBP)responsible for the activity is local-
ized in inner portion in oocyte and soluble
in aqueous solution(s-VBP). The role of s-
VBP was supposed to be vitellin condenser
holding high concentration of vitellin
in mature oocytes. Further purification of
s-VBP was carried out and could be purified
over 200 times compared with starting
material. A component of the purified s-VBP,
Fp was estimated to be an active binding
portion with vitellin. This fraction was
prepared by PAGE and electro-ellution.
FITC-Fs produced complex with Vn . Specific
binding of Fs with vitellin was clearly
shown by native PAGE analysis. The purific-
ation method for Fs involved complicated
procedure. However, the more simple prepar-
ation method for Fp, could be established by
the results of characterization,especially
behavior of the Fs for organic solvents.
It is estimated Fx, contains several compo-
nets active for Vn binding and have not
suitable category for the characteristics
as chemical substance.
Biochemistry 1151
IDENTIFICATION OF TETRAHYMENA 14-NM FILA-
MENT-ASSOCIATED PROTEIN AS ELONGATION
FACTOR la
Y.Kurasawa, O.Numata, M.Katoh, H.Hirano!
and Y.Watanabe. Inst. of Biol. Sci., Univ.
of Tsukuba, Tsukuba and !National Inst. of
Agrobiol. Resources, Tsukuba
Tetrahymena 14-nm filament-forming
protein has dual functions as a citrate
synthase in mitochondria, and as a cyto-
skeletal protein involved in oral morpho-
genesis and in pronuclear behavior during
conjugation. The 14-nm filament protein
fraction prepared by assembly and disas-—
sembly contained two 49-kDa proteins whose
isoelectric points were 8.0 and 9.0.
Monoclonal antibodies against the 14-nm
filament protein fraction were clearly
separated into two groups reacting to pI
8.0 or pI 9.0 protein specifically. From
the N-terminal amino acid sequence, the pI
8.0 protein was identified as the 14-nm
filament-forming protein. The pI 9.0
protein is considered to be a 14-nm fila-
ment-—associated protein since the pI 9.0
protein copurifies with the pI 8.0 protein
during two cycles of an assembly and
disassembly purification protocol.
Cloning and sequencing the pI 9.0 protein
gene from a T. pyriformis cDNA library, we
identified the pI 9.0 protein as elonga-—
tion factor la (EF-la) based on it sharing
73-76% sequence identity with EF-la from
several species.
PRIMARY STRUCTURE AND PROPOSED TERTIARY
STRUCTURE MODEL OF THE SQUID SODIUM
CHANNEL.
Chikara Sato, Kiyonori Hirota and Gen
Matsumoto. Electrotechnical Laboratory,
Supermolecular Science Divison, Tsukuba.
The complete amino acid sequence of a
sodium channel from squid Loligo bleekeri
has been deduced by cloning and sequence
analysis of the complementary DNA. The
deduced sequence revealed an organization
virtually identical to the vertebrate
sodium channel proteins; four homologous
domains containing all six membrane-
spanning structures are repeated in tandem
with connecting linkers of various sizes.
A unique feature of the squid Na channel is
the 1,522 residue sequence, approximately
three fourths of those of the rat sodium
channels I, II and III. On the basis of
the result, we have proposed a tertiary
structure model of the sodium channel where
the transmembrane segments are octagonally
aligned and the four linkers of S5-6
between segments S5 and S6 play a crucial
role in the activation gate, voltage sensor
and ion selective pore, which can slide,
depending on membrane potentials, along
inner walls consisting of segments S2 and
S4 alternately. The proposed model is
contrasted with that of Noda et al.. The
proposed model could explain various
unsolved phenomena, for example, effect of
TTX (tetrodotoxin) on the gating current of
the sodium channnel.
THE BINDING SITE FOR COLLAGEN FIBRILS ON A
CELLULAR FIBRONECTIN (cFN) MOLECULE
M. TSukahara, K. Yoshizato
Moi. (Celli) Scr. (Zab. ,, Zool Inst. , Face of
Sci., Hiroshima Univ., Higashihiroshima
The collagen gel culture is a useful
experimental system in which the mode of
binding between fibroblasts (Fbs) and
collagen fibrils (CL) can be analyzed. With
this culture Fbs recognize CL through cFN
but not plasma FN (pFN). We have obtained
monoclonal antibody (A3A5) which inhibits
human Fbs-mediated collagen gel contraction
and recognizes human FN. Quantitative
assays of the extent of Fbs spreading on CL
also suggested the involvement of cFN in
binding of Fbs to CL. The region in FN
recognized by A3A5 was analyzed. Human pFN
was digested with trypsin and was subjected
to immunoblot analyses with A3A5. The
shortest fragment with molecular weight
19.5 kDa which reacts with A3A5 was
analyzed for the to amino acid sequence and
was found to have a sequence of TAGPDQT at
the N-terminal region. From this we suppose
that the binding site of CL on cFN is
localized between typeIII region on the N-
terminal side of ED-A and the middle of
Heparin domein. For more precise analyses
on the recognition site of A3A5 were
carried out using human FN cDNAs encoding
cFN and pFN. FN cDNAs fragments contained
resion of 19.5 kDa trypsin fragment were
constructed in vector and expressed as a
fusion protein. This protein is now under
investigation for the reactivity with A3A5.
BREAKDOWN OF LARVAL TISSUES OF AMPHIBIAN
TADPOLE BY ASPARTIC PROTEINASE.
M. Mukai, K. Yoshizato, Mol. Cell Sci.
iets), “Aol AGNCigs., ICs Weise Whehhirg = Cpr
Hiroshima, Higashi-hiroshima.
Amphibian tadpoles undergo metamorphosis
and convert themselves into frogs by
destroying their larval tissues and
developing adult ones. The activity of
acidic proteinases increases markedly in
metamorphosing tail tissue. They are one of
key enzymes in this histolysis.
This study examined the activity of
aspartic proteinase in tissues of tail,
limbs and dorsal body muscle of
metamorphosing bullfrog tadpole ( Rana
catesbaiana) in relation to changes in DNA
contents. The results suggested that the
process of tissue degradation is divided
into two phases. In the first phase
activity of aspartic proteinase increased
which was observed in both tail and dorsal
body muscle at Taylor and Kollros stage
XXI. This phase did not show any changes in
the DNA contents per tissue weights. In the
second phase which was found only in tail
tissue (especially strong in distal part of
tail) at T-K stage XXII, the activity
further increased with a concomitant rise
of DNA content which is most probable due
to tissue condensation. The first phase may
reflect autolysis, while the second is
heterolysis probably caused by invading
Macrophages. The enzyme activity in limbs
remained quite low during metamorphosis.
1152 Biochemistry
BILIVERDIN-ASSOCIATED CYANOPROTEIN OF RIP-
TORTUS CLAVATUS MOLECULAR CLONING, SE-
QUENCE ANALYSIS AND REGULATION BY JUVENILE
HORMONE.
1K. Miura, 2T. Shinoda, 1M. Yura, 1S. Nomura and
ly. Chinzei. ‘Dep. Med. Zool., Mie Univ., Tsu, and
2Natl. Inst. for Tea, Mie Pref.
Biliverdin-associated cyanoproteins of the bean
bug, Riptortus clavatus (CP-1 to 4) are hexameric
proteins composed of two different subunit, CPa@
and CPZ. The cDNA libraries were constructed
from the fat body polyA(+) RNA of the reproduc-
tive and diapause females. cDNA clones encoding
CPa and CP8 were screened from the cDNA li-
braries by using anti-CP-1 and anti-CP-4 sera.
The nucleotide sequences of the CPa and CPB
cDNAs predicted that CPa and CP8 contain 693
and 686 amino acid residues, respectively, and the
identity at the level of amino acid sequence be-
tween the two subunit calculated to be 68%.
Homology search revealed that the cyanoprotein
falls into ‘hexamerin’ superfamily which includes
insect storage hexamers and arthropod hemocya-
nins. The expression of the cyanoproteins were
examined by northern blotting of the fat body
total RNA using the CPa and CP8 cDNA clones as
radioactive probes. Juvenile hormone (JH) treat-
ment to the diapause bugs resulted in complete
diapause termination. During the course of the
diapause termination the shift of CP expression
from CP8 to CPa@ was observed in the females
while the JH treatment suppressed completely the
expression of both CPa and CP in the diapause
males.
STRUCTURAL ANALYSIS OF INTERNAL TRANSCRIBED
SPACERS(ITS) AND 28SrDNA OF APHID.
D.Amako, O-Y.Kwon and H.Ishikawa.
Zool.Inst., Fac.Sci., Univ. of Tokyo, Tokyo.
In the process of forming mature 18SrRNA,
5.8SrRNA and 28SrRNA in eukaryotes, two
regions of the pre-rRNA are cut off through
post-transcriptional processing. One is ITS1
which is between 18SrRNA and 5.8SrRNA; the
other, ITS2 between 5.8SrRNA and 28SrRNA.
To characterize these regions of the pea
aphid, Acythosiphon pisum, we determined
their primary sequences and constructed
their presumable secondary structures. ITS1
and ITS2 of aphid consisted of 229 bases and
280 bases, whose G/C contents were 70% and
74% respectively. These G/C contents were
surprisingly high compared with other lower
invertebrates. The aphid ITSs were rather
comparable to those of vertebrates. In
addition, it was shown that despite
extensive divergence in nucleotide sequence
and G/C content, the secondary structures of
ITSs are conserved throughout species. We
also determined the partial sequence of the
aphid 28SrDNA which is exceptional in that
its molecular weight is larger than other
organisms by 200KDa, and that it does not
contain "hidden break". So far about 3,000
bases have been sequenced and the followings
were suggested: 1) G/C content was
approximate 60%, 2) Not so similar to those
of other organisms, 3) Highly conserved
regions were separated from each other by
long non-conserved regions.
STUDIES ON THE MECHANISM OF FLIGHT MUSCLE
BREAKDOWN IN APHIDS.
M. Kobayashi and H. Ishikawa. Zool. Inst.,
Fac. of Sci., University of Tokyo, Tokyo.
We studied flight muscle breakdown of
the pea aphids, Acyrthosiphon pisum
(Harris), in relation to their flight,
feeding and reproductive behavior. Using
the acetone-freeze dry microdissection
technique, which enables us to isolate the
indirect flight muscle (IFM) free from
other tissues without proteolysis, we
monitored its development and breakdown in
terms of its protein content. IFM protein
content reached the maximum on the 2nd day
after the final ecdysis, and then turned
to decrease. Histological studies indicat-
ed this decline is due to the breakdown of
IFM. Intriguingly, body weight of alatae,
unlike that of apterae, decreased during
the first 2 days after the final ecdysis
in inverse proportion to IFM development,
thus providing the suitable conditions for
flight. In addition, body weight turned to
increase coincidentally with the onset of
IFM breakdown, followed by larviposition,
suggesting that fecoing not only provides
raw materials for producing progeny but
also serves as certain stimuli to bring
about muscle breakdown. In fact, in the
starved aphids, neither the IFM breakdown
nor reproduction took place until these
insects were fed later. The fact that the
administration of cycloheximide prevented
IFM breakdown indicated feeding stimulates
de novo syntheses of certain proteins
essential to this process.
AMINO ACID EXCRETION OF ENDOSYMBIOTIC
SYSTEM OF APHIDS.
T. Sasaki and H. Ishikawa. Zool. Inst., Fac. Sci., Univ. of
Tokyo, Tokyo.
Bacterial endosymbionts of aphids have been suggested to
play important roles in the nitrogen metabolism of the host
insects. We maintained symbiotic and aposymbiotic pea
aphids, Acyrthosiphon pisum on a chemically defined syn-
thetic diet, and analyzed nitrogen compounds in the honeydew
excreted by the aphids. Unlike most insects, aphids did not ex-
crete uric acid as a nitrogenous waste but instead amino acids.
The principal amino acid in the honeydew of aposymbiotic
aphids was glutamine, while symbiotic aphids scarcely ex-
creted it. The amino acids in the honeydew are a mixture of
metabolic products and those passed through the gut without
being absorbed or subjected to metabolic manipulation. To tell
one from the other, aposymbiotic aphids were kept on a diet
from which glutamine was omitted. Even on such a diet, they
excreted appreciable amount of glutamine, confirming that they
excreted glutamine as a nitrogenous waste. For a further study,
aposymbiotic aphids were kept on a diet containing [e-
1SN]glutamine. In their honeydew, asparagine was found
labeled. We also found an unknown ninhydnn-positive spot on
TLC which was highly labeled with 15N. Analyses by NMR
and GC-MAS revealed that this spot is due to a mixture of
y-glutamylglutamine and y-glutamylasparagine. It was sug-
gested that aposymbiotic aphids excrete amides and y-
glutamylamides, while these amides are reutilized by the
symbiotic aphids, possibly with the aid of their endosym-
bionts.
Biochemistry 1153
PHOSPHORYLATION OF SYMBIONIN.
M. Morioka and H. Ishikawa. JZool.Inst.,
Fac.Sci., Univ.of Tokyo, Tokyo.
Symbionin, a GroEL-homologous heat shock
protein of aphid endosymbiont, functions as
molecular chaperone in folding and assem-
bling polypeptide in vitro. Also, symbionin
is autocatalytically phosphorylated in
vitro in response to the temperature shift-
up. In this paper, we report the
biochemical significance of phosphorylation
of symbionin as a molecular chaperone.
When the chemically unfolded dihydro-
forate reductase (u-DHFR) was diluted in
refolding buffer, the spontaneous refolding
of u-DHFR was strongly inhibited by GroEL
but not by symbionin. By contrast, the
refolding of u-DHFR was inhibited by the
phosphorylated symbionin, suggesting that
the latent domain contributing for the
chaperonin activity of symbionin is
activated by the phosphorylation. The
latent chaperonin activity of symbionin was
also activated with several kinds of salt.
In addition, when the 1M NaCl-treated
symbionin was subjected to native PAGE or
gel-filtration, 70kDa-protein was separated
from symbionin, suggesting that symbionin
exists as a complex with 70kDa-symbionin
binding protein, which plays an important
role as a regulatory factor of molecular
chaperone in the endosymbiont.
PHOSPHOTRANSFERASE ACTIVITY SPECIFIC TO
SYMBIONIN, A GROEL PROTEIN HOMOLOGUE.
H.Muraoka, M.Morioka and H.Ishikawa.
Zool.Inst.,Fac.Sci.,Univ.of Tokyo, Tokyo.
EE ee ee eee
Endosymbionts, when habored by the aphid
bacteriocyte, selectively synthesize
symbionin, a structual homologue of the
GroEL protein, a heat shock protein of
E.coli. Isolated endosymbionts under a
temparature shift-up synthesize the 63KDa
heat shock protein which is a
phosphorylated form of symbionin.
In this study, we showed that symbionin has
auto-kinase activity, and that the
phophorylated symbionin is able to transfer
its phosphate to ADP. It was also shown
that symbionin transfers phosphate group
from ATP to GDP, while that the GroEL
protein has ATPase activity but is the
apparently without transferase activity.
These results suggested that the
phosphorylated symbionin has a high energy
bond and serves as a high-energy phosphate
donor. Therefore, it is likely that
symbionin has other functions in addition
to chaperonin activity common to the GroEL
protein.
1154 Developmental Biology
GRAFTS OF CENTER CELLS INTO EMBRYOS AT
DIFFERENT STAGES IN HORSESHOE CRABS.
T. ITOW. Dept.of Biol., Fac.of Edu.,
Shizuoka Univ., Shizuoka.
The center cells under the blastopore of
horseshoe crab embryos at early gastula
stage induce secondary embryos after the
grafting of the center cells into embryos at
the same stage.
Fot the understanding of characteristics
of embryonic induction by center cells, the
center cells of horse shoe crab embryos. at
early gastrula stage were grafted into the
embryos at the stages earlier than _ early
gastrula. “Then the center cells of early
gastrula were grafted into the embryos at the
stages later than early gastrula. As the
results of both grafts, the secondary embryos
were not formed, and the treated embryos
developed normally. Those facts mean that
the center cells at early gastrula can not
induce secondary embryos at the stages
different from early gastrula.
Besides, the center cells of embryos at
the stage later than early gastrula were
grafted into the embryos at early gastula
stage. As the results of those grafts,
secondary embryos were induced. The fact
means that the center cells of embryos after
early gastrula stage remain the ability of
embryonic induction.
ANALYSIS OF AMINO ACID - LIKE CHROMATIN
COMPONENTS OF EARLY NEWT EMBRYOS
T. Asao, Biol. Lab., Sch. Med., St.Marianna
Univ., Kawasaki
Amino acid-like components in the nuclear
extract of newt embryos were analyzed by
gel, ion exchange and reverse phase chroma-—
tography. The results from chromatographic
profiles showed, firstly, that these were a
group of substances of similar molecular
weight below several hundred daltons and
were released from the nucleus most effi-
ciently with around a 50 to 100 mM NaCl
solution. Secondly, they were composed of
three kinds of group, i.e.,hydrophilic, in-
termediate and less hydrophilic substances
showing amino acid properties, the last
group of which increased as development
proceeded from blastula to tail bud stage.
Thirdly, the regional differences in the
chromatographic profiles were noted among
the anterior and posterior neural plates
and the epidermis in neurula. Reverse phase
chromatography showed that medium rearing
gastrula tissue fragments for a day con-
tained several substances similar to nucle-
ar extract components. An infra red spec-
trum of the specimens recovered from gel
chromatography, however, was similar to
that of fructose. Furthermore, the physio-
logical activity in vivo of the specimens
above was hardly positive. These unfavor-
able results are thought to be due to the
incomplete purification of the substances.
DORSAL CYTOPLASMIC TRANSFER INTO XENOPUS EARLY
EMBRYOS DEVELOPED FROM UV-IRRADIATED-FERTILIZED
EGGS OR -OOCYTES.
N. Mise and M. Wakahara, Zool. Inst., Fac. Sci. Hokkaido Univ.,
Sapporo.
Previous works have shown that ultraviolet (UV) irradiation to
vegetal region of fertilized eggs (UV-F) and oocytes (UV-O) of
Xenopus laevis yields embryos that lack dorsal axial structures
(dorsal axial structure deficiency) in the same way, but UV targets
are thought to be different respectively. On the other hand, it has
been shown that cytoplasm of dorsa! vegetal blastomeres of early
embryo (dorsal cytoplasm) contains "dorsal determinants”.
UV-F embryos had circular blastopores, and they did not have
dorso-ventral polarity in appearance. UV—O embryos had normal
blastopores with clear dorso-ventral polarity in early gastrula stage.
Injecting dorsal cytoplasm from 8-16 cell stage embryos could
rescue the dorsal axial structure deficiency of UV-F embryos. But
we could not rescue UV—-O embryos by cytoplasmic transfer. These
results suggest that the establishment of dorso-ventral polarity and
the development of dorsal axial structures should be analyzed
separately.
EXPRESSION OF THE EMBRYONIC EPIDERMAL
DIFFERENTIATION-SPECIFIC TRANSCRIPT IN
ADULT TISSUES OF THE JAPANESE NEWT, CYNOPS
PYRRHOGASTER.
T.C.Takahashi and K.Takeshima,
Radioisotope Research Center, Nagoya
University, Nagoya 464-01 JAPAN
Embryonic epidermal differentiation-
specific cDNA isolated from Cynops neurula
library (Develop.Growth&Differ., 34, 277-
283, 1992) was used for studying its gene
expression both in embryos and in adult
tissues. The HindIII-SphI fragment of the
cDNA, pG3m11N138-48, was prepared for the
probe for Northern hybridization analyses.
When RNAs were isolated from the whole
embryos of various developmental stages,
the Message was first detected at stage
15, the stage characterized by the
disappearance of the yolk plug prior to
the neural plate formation. The message
was then increased and was very abundant
throughout the larval stages. When RNAs
were purified from adult tissues, the
message was not abundant and detected by
less intensity. Interesting result was
the appearance of different size of the
transcript in adult tissues when compared
to the size from larval and embryonic
preparation. These results suggests that
the expression of this gene is regulated
in time and in space not only during the
early development but also in post-
metamorphotic growth and differentiation.
Developmental Biology 1155
CON A-LIKE MOLECULES SPECIFIC TO THE ORGA-
NIZER AND THE DORSALIZED VENTRAL MESODERM
MRM, Wehey_p Mss Takata and S. Takesue-.
1 Nagoya University, College of Medical
Technology, Higashi-ku, Nagoya 461
Doho University, General Education,
Nakamura-ku, Nagoya.
- Con A-treated ventral mesoderm was found to devel—
op notochord and muscle tissues typical of the orga-
nizer and was found to attain peak neural-inducing
activity 24h after treatment (Diazetal., 1991). This
time, at the protein level, wewere able to showthat a
couple of bands were specific to both the organizer
and the 24h-old dorsalized ventral mesoderm.
Organizer samples were analyzed by SDS polyac-
rylamide gel electrophoresis followed by immuno-
blotting using anti-Con A as probe. A
Several anti-Con A reactive proteins were de-
tected. Compared with immunoblots of the
endoderm and the ventral mesoderm, a few of
those bands appeared to be specific to the
organizer. Compared with the banding pattern
of the 24h-old dorsalized ventral mesoderm, a
couple of bands appeared to be common with the
Organizer, but not with the untreated ventral
mesoderm.
These results offer additional evidence to the possi-
bility that the organizer is aCon A-like substance.
More detailed research onthe nature of the organizer
at the molecular level may be approached using Con
A system as the working model.
INDUCING ABILITIES OF THE XENOPUS AND
CYNOPS PRESUMPTIVE ECTODERM TREATED WITH
ACTIVIN A.
T.Ariizumi, Z.Hasegawa and M.Asashima.
Dept. of Biol., Yokohama City Univ., Seto,
Kanazawa, Yokohama 236.
We examined the inducing abilities of
the presumptive ectoderm of early amphibian
embryos after treatment with activin A. We
treated the ectoderm of late Xenopus
blastula with 10 - 100 ng/ml of activin A
for 10 minutes and "precultured" it for 0 -
7 hours. The treated ectoderm was
sandwiched between two sheets of ectoderm
from another late blastula embryos. Short-
precultured ectoderm induced trunk-tail
Structures, whereas long-precultured
ectoderm induced head structures. These
changes of inducing abilities, dependent
upon the time of preculture, occurred more
rapidly when the concentration of activin A
was higher. Similar phenomenon was observed
in the experiments using Cynops embryos. In
addition, beating heart was also induced in
the Cynops explant. These results suggest
that the activin A-treated ectoderm behaves
like the Spemann's organizer and functions
as “head organizer" or "“trunk-tail
organizer", depending upon two factors: the
concentration of activin A, and the time of
preculture.
INDUCTION OF THE ANIMAL BLASTOMERES
OF THE 8-CELL EMBRYO OF XENOPUS
LAEVIS BY ACTIVIN A.
T. Bessho, A. Fukui, H. Uchiyama and
M. Asashima. Dept. of Biol., Yokohama City
Univ., Yokohama 236.
Xenopus blastula ectoderm is induced to
mesoderm by activin A. However, maternal
activin is presence in the unfertilized
egg, and the real mesoderm induction is
considered to occur earlier. We separated
a single animal blastomere from 8-cell
embryo, and at once treated it with 50
ng/ml of activin A for 1 h. The dorsal
blastomere mainly formed notochord and
neural tissues, in contrast to the ventral
one which formed mainly mesenchyme and
coelomic epithelium or atypical epidermis.
Next, we precultured the blastomere in a
saline for 1 or 2 h before treatment with
activin. We had same results as the above
experiment. When the blastomere is treated
first with activin for 1 h, followed by
follistatin at 500 ng/ml for 4 days, the
mesodermal induction was observed. Respon-
siveness of dorsal and ventral blastomeres
to activin A are different. These results
suggest that activin receptor exist in the
animal blastomeres of the 8-cell embryo.
MESODERM-INDUCING ACTIVITY OF INHIBINS IN
XENOPUS EMBRYOS.
H.Nakano', H.Sugino2, and M.Asashima'. ‘Dept. of
Biol., Yokohama City Univ., Yokohama, @Inst. for
Enzyme Res., Tokushima Univ., Tokushima.
We previously reported about high mesoderm-inducing
activity of activin A, and lower activity of inhibin than that
of activin A. An activin A molecule is a homodimeric
protein composed of two inhibin B,—-subunit chains. An
inhibin molecule, heterodimeric protein consisted of a-
and B-subunits, has two isoform types, such as inhibin A
(a + B,) and inhibin B (a + Bg). Mesoderm—inducing
activity of those inhibin A and B was tested by animal
cap assay method using Xenopus laevis embryo. In the
cases of both inhibin A and B, mesoderm induction
accompanying with mesenchyme and coelomic epitheli-
um was recognized at a concentration of 100 ng/ml, but
not at of 10 ng/ml. The concentration of inhibins needed
for the mesoderm induction is higher than that of activin
A. To test the combination effect of inhibin A or B with
activin A, activin A with inhibin A or B were co-incubated
before bioassay. Then assay was carried out. As a re-
sult, mesoderm-induction was reduced a bit, and per—
centages of induced tissue types were slightly changed,
respectively. Inducing activity and effect on induction of
a-subunit monomers (26 and 46 forms) were also tested
by the same bioassay method. The relationships be-
tween composition forms of these proteins and meso-
derm-inducing activity are discussed.
1156 Developmental Biology
PURIFICATION OF XENOPUS ACTIVINS A, AB AND
B, FROM CONDITIONED MEDIUM OF XTC CELLS.
A. Fukuil, re Nakamura2, H. Uchiyamat, Be
Sugino2 and M. Asashimat.
1pept. of Biology, Yokohama City Univ.,
Yokohama 236, Inst. for Enzyme Res.,
Tokushima Univ., Tokushima 770.
Activin is composed of two inhibin 6
chain subunits. Activin A, AB and B
isoforms are composed of fafa homodimer,
Babe heterodimer and phy homodimer,
respectively. Follistatin, an activin-
binding protein, suppresses physiological
activities of activins. We have purified
Xenopus follistatin, and activins A, AB and
B, from conditioned medium of XTC cells
using four steps of column chromatography.
The activins bounded with follistatin in
the conditioned medium. The Activin-
follistatin complex was separated by the
last reverse phase HPLC, into follistatin
and Activins, A, AB and B. The identities
of these molecules were revealed by SDS-
PAGE and immunoblotting. Xenopus activins
were determined by erythroid differentia-
tion activity (EDF assay) using porcine
activins as standard. Xenopus activins
induced mesodermal tissues from ventral to
dorsal ones, in dose-dependently.
OVER EXPRESSION OF FOLLISTATIN mRNA IN THE
EARLY EMBRYO OF XENOPUS LAEVIS .
M.Iwao, S.Shimazaki*, S.Nishimatsu**,
N.Ueno**, A.Fukui, H.Uchiyama and
M.Asashima. Dept.of Biol., Yokohama City
Univ., yokohama; *Whittier Inst., San Diego,
USA.; **Inst. of Appl. Biochem., Tsukuba
Univ., Tsukuba.
Activin, a Xenopus oocyte's maternal
protein, has the mesodermal inducinhg
activity on isolated early Xenopus animal
caps. This inducing activity is inhibited in
vitro by follistatin, an activin-specific
binding protein. To investigate the activin
function in vivo, we injected follistatin
mRNA into blastomeres at St.3. The injected
embryos delayed the closure of the
blastopore, and had abnormal eyes. Many of
them were small or lensless. On the other
hand, some embryos had the third cement
gland and the eye on their forehead. The
ratio of the abnormal embryo was increased
dose-dependently with the amounts of
injected mRNA. When follistatin mRNA was
injected with activin mRNA, they
counteracted each other. This suggests that
injected mRNA was translated, and that the
products had the activity in vivo.
Therefore, the effect of injected
follistatin mRNA seems to be inhibit native
activn activty. Translation of injected
follistatin mRNA began at early gastrula,
and decreesed from early neurula stage.
Therefore, the mRNA of this follistatin was
not translated during mesodermal induction
(St.6=7) .
MESODERM INDUCTION BY ACTIVIN A ON THE
XENOPUS DIVIDING STAGE ANIMAL HALF CELLS.
K.Kinoshita!, T.Kinoshita’?, M.Asashima?.
1Biol. Lab., Nippon Med. School, Kawasaki,
2Z001. Inst., Hiroshima Univ.,
*Dept. of Biology., Yokohama City Univ.
Activins act on the stage 8 animal cap
cells of Xenopus embryo to induce mesoder-
mal tissues. However, we know little about
when animal half cells become acceptable
for activins. We isolated animal half
blastomeres at early dividing stage (after
st.4), then treated with 50 ng/ml activin
A. RNAase protection assay showed that
muscle a-actin mRNA was present in the
explants derived from the blastomeres
treated with activin at the onset of st.5.
The level of mRNA remarkably increased
following the stage of treatment. It was
synthesized mostly in the future dorsal
cells. When activin was treated on the
cells of st.6 embryos, no evident differ-
ence was observed between the cells of
animal pole and dorsal marginal zone.
The responsiveness to activin was also
estimated by goosecoid gene, an organizer
specific homeobox gene. The goosecoid mRNA
was transcribed soon after activin treat-
ment in st.8 animal caps. The timing of
goosecoid expression was not influenced by
the activin treatment at st.5.
The results suggested that dorsal animal
half cells of stage 5 possess some activin
receptors and the timing of goosecoid gene
expression seems to be regulated indepen-
dently of the stage of mesoderm induction.
AXIS FORMATION AND THE EXPRESSION OF A
HOMEOBOX GENE IN EARLY XENOPUS EMBRYOS.
Koichiro Shiokawal, Misaki Asanol, Yasuko
Sugase~, Yasufumi Emori*, Kaoru Saigo4,
Lab. Mol. Embryol., Zool. Inst., “Dent.
Biophys. Biochem., Univ. Tokyo, Tokyo.
In animal embryogenesis, various homeo-
box genes and growth factors have been
implicated as important factors responsi-
ble for embryo axis formation.
We have recently isolated a novel homeo-
box gene of Xenopus laevis from the ovary
cDNA library (Asano et al.: J. Biol. Chem.,
267, 5044-5047, 1992). The homeodomain of
the gene was homologous to that of Droso-
hila Distal-less, and the gene was named
Tl. The mRNA exists in the unfertilized
egg’as maternal mRNA, and the level of the
mRNA was constant during the cleavage to
the early blastula, but increases from the
blastula to gastrula stage.
Embryo dissection experiments reveal-
ed that the Xdll mRNA is most abundant in
the head part, especially in the cement
gland, and its amount is much smaller in
the trunk and tail regions, The relative
abundance of the mRNA coincides the antero-
posterior axis, and the direction of the
invagination of the mesodermal cells during
the gastrula stage.
These results suggest strongly that the
Xdll mRNA may play an important role in
determining the head to tail or antero-
posterior axis in Xenopus embryogenesis.
Developmental Biology 1157
SEA URCHIN HATCHING ENZYME: THE SUGGESTED "SECOND
ENZYME" IS POSSIBLY A SMALL FORM AUTOLYTICALLY
DERIVED FROM ENVELYSIN
K. Nomura!, and N. Suzuki2, !Dept. of Biochenm.,
okyo Metropol. Inst. of Gerontol., Tokyo, and
Noto Marine Lab., Kanazawa Univ., Uchiura
The sea urchin hatching enzyme (HEz) belongs
to the mammalian matrix metalloproteinase (MMP)
family!,2 We have isolated from Hemicentrotus
pulcherrimus a 37 kDa HEz, designated "envelysin",
which alone is able to dissolve the fertilization
envelope (FE). A possibility has been suggested
that another enzyme is involved in the hatching
process. Indeed, we found during the Red-agarose
chromatography a fraction with caseinolytic
activity but without hatching enzyme activity .
We made a hypothesis that the enzyme is an auto-
catalytic derivative of envelysin by truncation of
its C-terminal domain having a role of binding to
FE, the specific substrate. Supporting evidences
are shown below. Envelysin, left at 27 °C for 13 h,
autodegrades into 32 kDa form with drastically
reduced hatching activity. However it retained
almost full caseinolytic activity as well as full
activity on (Xj j-antitrypsin. The caseinolytic
fraction without hatching activity, that did not
bind to the Red-agarose column, was inhibited by
EDTA and the cysteine-switch peptide, typical
inhibitors of envelysin. This enzyme seems to
be distinct from the 64 kDa protease homologous to
the human BMP-15. Experiments from protein
chemical and immunological aspects are now in
HOR RESS to further prove the above hypothesis.
EMBO J. 9 3003 (1990). 2Biochem. 30 6115 (1991).
SDevelopment 114 147 (1992).
Analysis of binding action of HCE, a con-
stituent of the hatching enzyme of medaka,
toward chorion
Lee K., S.Yasumasu, I.Iuchi, K.Yamagami
Life Sci. Inst., Sophia Univ., Tokyo
The hatching enzyme of medaka, Oryzias
latipes, consists of two enzymes, HCE and
LCE. HCE swells the inner layer of chorion
by hydrolyzing it partially and LCE
hydrolyzes the swollen part of the inner
layer. HCE tends to bind tightly to the
chorion when it causes the chorion swell-
ing. Present study examined by SDS-PAGE
whether the enzyme binds to chorion
EUSMElYy DieiOGrm CO CloOri@lwSiss A
monoclonal anti-HCE antibody(E72) inhibits
the choriolytic activity of HCE, but it
does not affect the proteolytic activity
of the enzyme. But how E72 inhibits the
choriolysis of HCE is not well known.
When a definite amount of HCE was prein-
cubated with varied amounts of E72, the
binding of the HCE to the chorion
decreased depending on the amount of the
E72. This result strongly suggests an in-
hibition of HCE binding to the chorion by
aU Z < Wows le LS COMPAiPMGC wWAAwB wae
choriolysis of HCE premises binding of
the enzyme toward the chorion. HCE
bound to the egg envelope of rainbow trout
but not to insoluble collagen. The binding
to the former was also inhibited by MAB
B2e
The Gene Structure of HCE, a Constituent
of the Hatching Enzyme of Medaka, Oryzias
latipes
S.Yasumasu7, K.Inohaya* K.Yamada?,
I.Iuchit+, H.Shimada? and K.Yamagamit
1: Life Sci. Inst., Sophia Univ. 2: Zool
Inst., Hiroshima Univ.
The hatching enzyme consists of two types
of proteases (HCE, LCE). In cloning of the
cDNA for HCE, two distinct cDNAs (HCE21,
HCE23) having nucleotide sequences with
92.8% similarity were obtained.
The genes encoding HCE, corresponding to
HCE23 (heel) and HCE21 (hce2), were iso-
lated from the genomic libraries con-
structed from the drR strain of medaka.
The structure analysis indicated that both
heel and hce2 contained the entire coding
sequences (approx 900bp), but lacked in-
trons. At least thee or four of hcel and
hce2 were tandemly repeated. The putative
TATA box consensus sequences were located
at about 50 bp upstreams from protein
coding regions. Their 5'flanking regions
locating in the upper stream of the
protein coding regions (200-400bp long)
highly resembled each other.
Comparison of gene structures of HCE and
LCE indicated that HCE gene was quite dif-
ferent from LCE gene, which contained 8
exons and 7 introns. HCE gene (hcel) was
located in about 8kb down stream of LCE
gene.
SOME PROPERTIES OF ERYTHROCYTE TRANSITION
DURING POST-HATCHING DEVELOPMENT OF RAINBOW
TROUT, Oncorhynchus mykiss.
I. Iuchi (Life Science Institute, Sophia
University, Tokyo)
It is well known that larval type eryth-
rocytes containing larval type hemoglobins
develop first on 7-8th day after fertiliza-
tion (15°C) and their replacement by adult
type erythrocytes containing adult type
hemoglobins occurs after hatching (Iuchi,
Zool., Sci., 2, 11-23, 1985). Treatment of
phenylhydrazine (10° °M) for 6-12 hr from
8th to 9th day after fertilization resulted
in degradation of larval type erythrocytes.
Embryos lacking almost all the erythrocytes
thus prepeared developed normally and
hatched out. They recovered neither larval
type erythrocytes nor adult type eryth-
rocytes, while they developed adult type
erythrocytes after hatching. This recovery
is ascribable to production of adult type
erythrocytes in the way similar to that in
non-treated alevins. Lack of larval type
erythrocytes did not affect the production
or differentiation of adult type eryth-
rocytes. On the other hand, X-ray irradia-
tion (2,000R) suppressed the production of
adult type erythrocytes. These results sug-
gest that the erythrocyte transition is not
easily influenced by extrinsic factors but
determined by some intrinsic constraints.
1158 Developmental Biology
LOCALIZATION OF M PHASE CELLS IN SEA
URCHIN BLASTULAE.
H.Mizoguchi, Lab. of Biol., Jun. Col. of Rissho
Univ., Saitama.
Propidium iodide was known to stain nuclear DNA.
We can infer M phase cells from the profile of
propidium iodide stained nuclei. Hence, in the
propidium iodide stained cells of sea urchin embryo at
the blastula stage, cell proliferation study was
performed to clarify the cellular mechanism of
archenteron formation.
Staining of the embryos by propidium iodide(0.5 uz
g/ml) was performed at the swimming blastula
stage( 11 hrs and 14hrs after fertilization at 18°C ) and
mesenchyme blastula stage(17 hrs and 20 hrs after
fertilization at 18°C ). Observations were performed by
fluorescence microscope. M phase cells were found in
the embryo uniformly at the swimming blastula stage.
Most of the M phase cells were localized at vegetalized
hemisphere at the mesenchyme blastula stage.
Percentage of M phase cells to the total cell number of
the embryo at the mesenchyme blastula stage is
greater than that of the embryo at the swimming
blastula stage.
Changes in frequency of M phase cells in the
embryo at the mesenchyme blastula stage may relate to
archenteron formation.
ARCHENTERON-FORMING ABILITY OF VEGETAL
REGION OF STARFISH EGG (3).
M.Kiyomoto and H.Shirai. Ushimado Marine
Laboratory, Okayama Univ. Okayama.
Archenteron-forming ability is
localized in vegetal region in immature or
mature oocytes of the starfish, Asterina
pectinifera. Animal egg fragments, which
are known to develop into only so-called
permanent blastulae, fused with vegetal
fragments can form archentera. If vegetal
fragments have been labeled with RITC,
only these archentera are composed of
labeled cells.
Fertilized eggs, whose fertilization
membranes were removed, were fused with
RITC-labeled vegetal fragments at the
animal pole. Many of these eggs developed
into gastrulae with two archentera; one
was RITC-labeled and the other was non-
labeled. The former was considered to be
derived from fused vegetal fragment and
the latter from the original vegetal
region. This result showed that the
determinant as well as other cytoplasm of
the vegetal fragment remained through the
cleavage stage in the original place at
which the egg-fragment fusion had
occurred. Thus, whole eggs (non-excised
eggs with intact animal and vegetal
region) can be used as recipients of the
assay for the determinant,
EXPRESSION OF ALKALINE PHOSPHATASE IN
STARFISH LARVAE.
Ritsu Kuraishi and Kenzi Osanai. Mar. Biol. Stn. Asamushi,
Tohoku Univ., Aomori,
In larvae of Asterina pectinifera, the presumptive area
of esophagus first invaginates at the start of gastrulation.
Then those of the stomach and intestine are added to the
archeneron gradually after the early gastrula stage. To
check whether these areas differentiate into endoderm
simultaneously or separately, we examined expression of
alkaline phosphatase (AP), a reported endoderm marker,
histochemically from the start of gastrulation to the
bipinnaria stage.
As a result, the presumptive area of the esophagus
and anterior part of stomach (area I) became AP-positive
several hours after its involution (27 h), while that of the
rest of the digestive tract (posterior part of stomach and
intestine; area Il) remained AP-negative until the mouth-
formation stage (42 h). The presumptive area of mesoderm
tissues (that of the anterior coeloms and mesenchymes
at the tip of the archenteron and that of the posterior
coelom in the dorsal wall of the archenteron), on the
other hand, was AP-negative throughout the stages
examined.
We have reported that complete removal of the area |!
from immature oocytes forbids the larvae to gastrulate,
though at least a part of the area Il is preserved. In those
larvae, expression of AP was also inhibited. Iductive
effect from the area | seems to be necessary for both
involution and the following differentiation of the area Il.
MORPHOGENESIS IN SPICULE-REMOVED EMBRYOS
OF THE SEA_URCHIN.
K. Shimizul2, y. Nakajima? and M. Ikeda!
Dept. of Biol., Keio Univ.,Yokohama and
2Biofouling Project, ERATO, JRDC, Yokoha-
ma.
In order to investigate the role of
spicules in morphogenesis of pluteus
larvae, embryos of the sea urchin, Cle-
peaster japonicus, were treated with sea
water containing 10 mM hydrochloride for
15 seconds at various stages. Spicules
were completely removed from embryos in
the acidic sea water. Although the treat-
ment delayed development, tissue differen-
tiation of the treated embryos were not
affected. More than 60 % of embryos treat-
ed at the early gastrula stage normally
developed into pluteus larvae. Almost
embryos processed at the prism stage,
however, became pluteus larvae with abnor-
mal morphology; round shape with lobe-like
short arms containing extraordinary spi-
cules. The time required for beginning to
regenerate spicules increased as the
stages treated were advanced. Immunohisto-
chemical study using monoclonal antibody
P4 which recognizes the antigen in the
spicule forming cells revealed that pat-
terning of the spicule forming cells were
disordered in the treated embryos. These
results suggest that spicule formation is
essential for morphogenesis of pluteus
larvae and at the same time formation of
proper pattern of spicules requires inter-
action with other tissues in the embryos.
Developmental Biology 1159
CHANGE iN MOLECULAR FORM AND ACTIVITY OF
ACETYLCHOLINESTERASE DURING SPICULE
FORMATION IN SEA URCHIN EMBRYO.
K. Ohta, M. Sato, T. Nakazawa, Department
of Biology, Faculty of Science, Toho
University, Funabashi.
In this study, the enzyme activity and
molecular change of acetylcholinesterase
were examined during embryonic development
cf sea urchin, Hemicentrotus pulcherrimus.
The acetylcholinesterase activity was
detected originally in unfertilized egg,
remaining in the low level before gastrula
stage. At gastrula stage, two molecular
forms of acetylcholinesterase were detected
by polyacrylamide gel electrophoresis. The
molecular masses were estimated as 140 kD
and 300 kD, respectively. The appearance of
higher molecular enzyme was consistent with
the occurrence of a remarkable increase in
the enzyme activity. This increase was
sensitive to emetine or puromycin but not
to actinomycin D. No activity of
cholinesterase (butyryl) was detected
throughout the development.
The spicule elongation was suppressed
by cholinesterase inhibitor, eserine. The
suppression of spicule elongation was
rescued by incubation in normal sea water.
A remarkable increase in the activity of
acetylcholinesterase and differentiation in
the enzyme pattern wouid involve in the
spicule formation.
SPICULE FORMATION-INDUCING SUBSTANCE IN
BLASTOCOELIC FLUID OF SEA URCHIN BLASTULA
M.Kiyomoto!’, M.Maruoka? and J.Tsukahara?.
1Ushimado Marine Laboratory, Okayama
Univ., Okayama, *Dept. of Biol., Fac. of
Sci., Kagoshima Univ., Kagoshima.
We reported spicule formation-inducing
substance (SFIS) in blastocoelic fluid
(BCF) of sea urchin blastula. The
presence of SFIS in BCF can be assessed by
spicule formation in decendants of
isolated micromeres under the influence of
BCF. SFIS is effective interspecificaly
and is inactivated by trypsin.
BCF of Toxopneustes pileolus and
Hemicentrotus pulcherrimus was
fractionated by gel filtration (HPLC,
Waters Protein Pack 300). Activity of
SFIS of IT. pileolus was eluted as a broad
peak (M. W. ca. 1x10*), and that of H.
pulcherrimus, as two peaks (M. W. ca.
3x105 and 1x10*).
In ion exchange chromatography (HPLC,
DEAE-Toyopearl 650), SFIS activity of T.
pileolus was obtained in gradient elution
at NaCl concentrations of ca. 0.5 M and
0.8 M, although total activity was
markedly reduced. That of H.
pulcherrimus, at ca. 0.3 M and 0.5 M NaCl
in step-wise elution.
ROLES OF TGF-B SUPERFAMILY GENES IN
DIFFERENTIATION OF MICROMERES OF SEA
URCHIN EMBRYOS.
T. Kinoshita! and K. Kinoshita”. 1Zool.
Inst., Fac. of Sci., Hiroshima Univ.,
Hiroshima and “Biol. Lab., Nippon Med.
School, Kawasaki.
The micromeres of 16 cell stage sea
urchin embryos can be isolated and
cultured in vitro. However, some serum
factors or blastocoelic factors should be
included in the culture media for the
differentiation of spicule-forming cells.
In order to examine the ability of
several defined peptides to induce the
spicule formation, micromeres of
Hemicentrotus pulcherrimus were cultured
in the sterile sea water supplemented
with EGF, bFGF, TGF-B or activin A.
Among the four peptides tested, only
activin A possessed the inducing
activity, although the percentage of
cells induced was very low.
Using cDNA fragments of Xenopus
activin and BMPs as probes, homologues of
these genes were screened from the cDNA
library of gastrula stage sea urchin
embryos. Seven clones were selected
through the second screening and were
subcloned. Northern hybridization
analysis showed that mRNA of at least one
clone existed in oocytes and cleavage
stage embryos. The mRNA gradually
decreases in amount after gastrulation.
SPICULE FORMATION OF PRIMARY MESENCHYME
CELLS IN THE ISOLATED BAGS FROM SEA URCHIN
EMBRYOS
T. Iwase and T. Miki-Noumura, Department of
Biology, Ochanomizu Univ., Ohtsuka, Tokyo.
The formation of spicular rudiments
and the growth of spicules in the bags were
examined in the culture media. Based on the
method of Whiteley et al.(1980), we able to
isolate the bags from sea urchin embryos
(Clypeaster japonicus) before formation of
spicular rudiments. In the bags, the
mesenchyme cells were enclosed with basal
lamina lining blastocoel. The
number of mesenchyme cells in it was
changed with the embryo stage. After adding
epitherial cells to the bags, they were
cultured in the culture media with or
without 3% horse serum (HS).
The bags were cultured without the
epitherial cells showed no spicule growth;
in contrast, the bags surrounded by
reaggregated epitherial cells were
assembled into a swimming gastrula-like
structure having remarkable spicule growth.
The culture medium containing HS induced
higher rates in the formation of spicular
rudiments and the growth of spicules than
that containing no HS.
So it can be said that the presence of
epitherial cells are essential to induce
spicule formation in the bags of the sea
urchin embryos (Clypeaster japonicus).
1160 Developmental Biology
METAMORPHIC POTENCY OF THE AGGREGATES
DERIVED FROM MESOMERES ISOLATED FROM
16-CELL-STAGE EMBRYOS OF A SAND DOLLAR,
PERONELLA JAPONICA.
S. Amemiya, Misaki Marine Biological
Station, University of Tokyo, Kanagawa.
The developmental potency of the
aggregates derived from mesomeres isolated
from 16-cell-stage embryos of a sand
dollar, Peronella japonica, was examined.
The eggs whose fertilization membrane was
removed chemically by treating with
trypsin were cultured to 16-cell-stage in
Cat*t-free sea water. Sixteen mesomeres
isolated from the 16-cell-stage embryos
were placed into 10-ml conical glass tubes
containing 0.5 ml of culture medium (100
ug/ml streptomycin and 100 u penicillin/ml
in Jamarin sea water= JSW), followed by
gentle centrifugation in order to group
them together on the tube bottom. The
mesomeres formed an aggregate and
developed into an embryoid. Two days
later, the embryoids were removed into
larger glass tubes containing 2 ml JSW,
followed to culture for about 30 days
more. The embryoids developed into larvae
having most larval structures such as arm
rods, archenteron and pigment cells within
several days, then formed some adult
structures such as spines and test plates.
Finally, they formed lantern and tube feet
and metamorphosed into complete juvenile
sea urchins.
FORMATION OF THE ADULT RUDIMENT AND METAMO-
RPHOSIS OF SEA URCHIN BY THYROID HORMONES.
Y.Chino, M.Kanda, K.Yamasu, T.Suyemitsu and
K.Ishihara. Dept. of Regul. Biol., Fac. of
Sci., Saitama Univ., Urawa, Saitama.
During development of the sea urchin,the
adult rudiment develops within the larval
body and renders the larva competent for
metamorphosis. Exploring the molecular
mechanisms of larval development that lead
to metamorphosis in the sea urchin, we
found that thyroid hormone (TH) induces
both formation of the adult rudiment and
metamorphosis in the sea urchins. TH is
actually present in the larval body, and
we found that TH accumulates to a remark-
ably high level in 8-armed larvae before
metamorphosis, when larvae are reared in
the presence of food, namely, algae, that
contains TH. Removal of algae inhibits the
accumulation of TH and arrests the forma-
tion of adult rudiments, suggesting that
TH is obtained from algae. Metamorphosis
of competent larva can be induced by TH,
but differences from natural metamorphosis
are apparent.
We like to propose for metamorphosis of
sea urchin that it is better to understand
as a consecutive process that can be
divided into two stages; a preceding pro-
cess of gradual formation of adult rudiment
and absorption of the larval body and a
ensuing rapid collapse of the residual
larval body and appearance of adult struc-
tures.
EFFECTS OF TEMPERATURE AND Ca2* CHELATOR ON ATTACHMENT
OF HEAD AND TAIL RESORPTION OF ASCIDIAN LARVA.
Y. Sato and M. Morisawa
Misaki Marine Biological Station, Fac. of Sci., Univ. of
Tokyo. Kanagawa. EEE
After hatching, tadpole larva of ascidian, attaches
to the bottom and then starts metamorphosis ; the
transition from the larval to the adult stage
Stimulation of the attachment has been considered to be
the signal for the later morphogenetic process, such as
the resorption of the tail, loss of the outer cuticular
layer of the tunic, the rotation of the visceral organs.
As the first step to know the relationship between
head attachment and tail resorption, we first studied
the effect of temperature on both events in the larva of
the ascidian Ciona intestinalis. When the larvae were
incuvated at 4%, attachment and complete resorption did
not occur for more than 24 hours though both events
occurred at room temperature (230) within 5 hours after
hatching. When temperature was brought back to the
original condition, tail was resorbed rapidly without
attachment in all larvae. Rotation of visceral organs
and the formation of the stron were completed after tail
was resorbed and then attachment of larvae began. These
results suggest that larval attachment is not the
trigger for the tail resorption.
Then we investigated the role of Ca** on the process
of metamorphosis. When hatched larvae were incuvated in
the artificial sea water containing 10°°M BAPTA-AM to
chelate intracellular (Ca**, attachment of larvae did not
occur and the adhesive papillae remained intact at the
tip of tadpole head. However, tail was resorbed
completely suggesting that intracellular Ca** takes part
in the attachment, but not play a key role in the tail
resorption. .
The experiments using temperature shock and (Ca**
chelator suggest that attachment and tail resorption of
ascidian larva may be controlled by different mechanism
CHANGES IN ELECTROPHORETIC PATTERNS OF EGG
PROTEINS DURING FERTILIZATION IN Oryzias
latipes.
T. Kani-e, T. Iwamatsu and T. Hirabayashi
Dept. Biol., Aichi Univ. of Educ., Kariya
448 and Inst. of Biol. Sci., Univ. of
Tsukuba, Ibaraki 305.
Changes in the two-dimentional SDS elec-
trophoretic patterns of extracts of fertil-
izing Oryzias latipes eggs were surveyed.
A major protein stainable with a coomassie
brilliant blue R-250 in unfertilized eggs
began to dissappear a few minutes and be-
came undetectable 85 min after insemina-
tion, followed by appearance of three dif-
ferent proteins. within ten min (23°C).
On the other hand, unfertilized eggs which
were immersed .in 25% acetone-saline for 30
sec (26 C) and failed to exhibit exocyto-
sis by insemination, were homogenized in
to 100 min from the acetone-treatment, or
insemination immediately after the treat-
ment. The electrophoretic patterns in these
eggs were compared with those in normally
fertilized eggs. In acetone treated eggs.
only a small spot of slightly alkaline pro-
tein among three proteins described above
became also detectable a few minutes after
acetone-treatment regardless of insemina-
tion. These observations suggest that in
the medaka egg, the alkaline protein be-
comes detectable by acetone-treatment, ir-
respective of exocytosis, while other three
change in a close relation with exocytosis.
Developmental Biology 1161
1-METHYLADENINE PRODUCTION BY OVARIAN
FOLLICLE CELLS RESPONSIBLE FOR OOCYTE
MATURATION IN STARFISH
M. Mita. Dept. of Biochem., Teikyo Univ.
Sch. of Med., Tokyo.
Resumption of meiosis in starfish
oocytes is induced by l-methyladenine (1-
MA) produced by ovarian follicle cells
under the influence of a gonad-stimulating
substance (GSS). This study was under-
taken to estimate the concentration of 1-MA
within the ovary of Asterina pectinifera
after stimulation with GSS. After
incubation of an ovarian fragment with GSS
at concentrations of more than 0.1lmg/ml for
about 20 min, spawning started
simultaneously with germinal vesicle
breakdown (GVBD) and follicular envelop
breakdown (FEBD). About 5 min of treatment
with either 0.1 mg/ml GSS or 0.1 pM 1-MA
was sufficient for induction of oocyte
release. The amount of 1-MA produced by
the ovary increased as the GSS
concentration was raised, and the longer
the time treatment with GSS, the greater
was the amount of 1-MA produced. Also, 1-
MA production was markedly dependent on the
content of ovary. About 130 oocytes were
contained per mg wet weight of ovary and
each oocyte was surrounded by a follicular
envelop consisting of about 50 cells.
Therefore, the content of 1-MA was
calculated to reach 0.18 pM at 5 min after
0.1 mg/ml GSS treatment. These results
strongly suggest that follicle cells are
enough to produce 1-MA for oocyte
Maturation in starfish.
STARFISH OOCYTE P34C4C2 KINASE ASSOCIATED
WITH DETERGENT-RESISTANT CYTOSKELETONS.
K.Kishimoto, S.Hisanaga and T.Kishimoto.
Lab. of Cell and Dev. Biol., Fac. of Bio-
sciences, Tokyo Inst. of Tech. Yokohama.
Onset of mitosis or meiosis is induced
by activation of a serine/threonine kinase
mamed maturation Eaanoiag factor (MPF),
consisting of p34°9°* and cyclin B. Al-
though MPF was assumed to be a soluble fac-
tor, our and other's recent results suggest
its, at least, four distinct subcellular
states: freely soluble, microtubule-associ-
ated, detergent resistant cytoskeleton-
associated and chromosome-associated. To
resolve how MPF displays different subcel-
lular states, we studied and compared
properties of detergent soluble _(DS)- and
detergent insoluble (DI )-p34e¢e kinase.
When starfish oocytes were treated with a
buffer containing 0.5% Nonidet P-40, about
half of histone Hl kinase activity was
extracted and the remaining half was recov-
ered in.the Di-fraction. Although DS- and
DI-p34°9°* showed similar properties in the
binding to p135¥°* and anti-cyclin B, and
the behavior on gel filtration, vimentin
kinase activity was different. The ratio
of VME GE oy BASSE H1 phosphorylation by
DI-p34°°°* was about 3-5 times lower than
that of DS-p34°9°*, Sucl-affinity column
purification of DI-p34°°°* increased _vimen-
tin kinase to the level of DS-p34C9°¢.
These results suggest the association of a
Beoeas which Amina Lies) Heetsinteatin kinase of
p34cdC2, with D1-p34Cde2 |
BINDING OF CYCLIN B AND MAP MEDIATES THE
ASSOCIATION OF MPF WITH MICROTUBULE.
K.Ookata, Saris an Aga, H.Murofushi~,
T.Itoh“, H.Hotani*, E.Okumura, K.Tachibana
and T.Kishimoto. Lab. Cell Dev. Biol.,
Tokyo Inst. Technol., Yokohama, lDept.
ENCIISIS 6 Biochem., Univ. Tokyo, Tokyo, and
Dept. Biosci., Teikyo Univ., Utsunomiya.
The onset of M-phase is controlled by
MPF (Mepbgse promoting factor), a complex
of p34°%C* and cyclin B. We demonstrated
previously that a portion of the complex
associates with microtubules (MTs) and
accumulates on meiotic spindle of starfish
oocytes. To address the mechanism and the
role of this association in vitro, in the
present study we used MAP4, which is a
major and well characterized microtubule-
associated protein in mitotic HeLa cells.
The cdc2-cyclin B complex, which is
purified from starfish oocytes with sucl-
affinity column, coprecipitated with MTs
in the presence of MAP4. GST-cyclin B-
Sepharose 4B bound to whole MAP4, and
preferentially to bacterially-produced
"Pro-rich fragment" of MAP4. The dark
field microscopic observation revealed
that the phosphorylation of MAP4 by the
cede2-cyclin B complex increased the
instability of an individual MT. These
results indicate that the association of
the cdc2-cyclin B complex with MT is
mediated by the binding of cyclin B and
the "Pro-rich region" in the C-terminal
half of MAP4 and the associated complex
affects the MT dynamics.
DISTRIBUTION AND CHANGES OF cdc2
HOMOLOGUES DURING CELL CYCLE IN THE NEWT
EGGS.
K. Takahara, Y. Iwao, M. Yamashita* and
M. Nagahama Inst. Biol., Fac. Sci.,
Yamaguchi Univ., Yamaguchi. XLab. of
Reprod. Biol., Natl. Inst. for Basic
Biology, Okazaki.
The accessory sperm nuclei in the
vegetal hemisphere degenerate in the
physiologically polyspermic newt, Cynops
pyrrhogaster eggs, probably due to
exposure to a low level of M phase-
promoting factor (MPF) activity. We
investigated the distribution of cdc2
kinase (a catalytic subunit of MPF) and
its relative molecules with anti-PSTAIR-
sequence antibody. A PSTAIR-containing
protein (32kDa) was observed both in
eytoplasm and in germinal vesicles of full
grown oocytes. Three proteins, major
32kDa, minor 31kDa and 30kDa, were
detected in the unfertilized eggs, but
81kDa protein appeared only in high MPF
condition. The 30kDa protein may be cdk2
protein by cross-reaction with anti-
goldfish-edk2 antibody. The animal
hemisphere was abundant about 2 times in
32kDa protein, and 4 times in 31kDa and
30kDa proteins, compared with that in the
vegetal hemisphere. These results suggest
higher activity of MPF in the animal
hemisphere than in the vegetal hemisphere.
1162 Developmental Biology
CYCLIN B IN FISH OOCYTES: ITS APPEARANCE DURING
OOCYTE MATURATION AND IMPLICATIONS FOR THE
MECHANISM OF MPF ACTIVATION.
Y. Katsu, M. Yamashita, H. Kajiura and Y. Nagahama
Lab. of Reprod. Biol., Natl. Inst. for Basic Biology, Okazaki.
Changes in the two components of maturation-promoting
factor (MPF), p34°42 and cyclin B, during 17 @,208-DP-in-
duced oocyte maturation in goldfish were examined using
newly developed monoclonal antibodies against the C-
terminal of goldfish p34°4? and E. coli-produced goldfish
cyclin B. The C-terminal p34°4? antibody recognized a
35kDa protein in immature oocyte extracts and 35kDa and
34kDa proteins in extracts from oocytes undergoing germinal
vesicle breakdown (GVBD). Cyclin B was absent in imma-
ture oocytes and appeared at GVBD, coinciding exactly with
the appearance of the 34kDa protein. Furthermore, immuno-
precipitate experiments revealed that cyclin B formed a
complex with the preexisting p34‘? as soon as it appeared.
Introduction of E. coli-produced goldfish cyclin B into
immature oocytes induced MPF activation both in vivo and in
vitro. It is concluded that (1) 35kDa and 34kDa proteins are
an inactive and active form of p34°4*, respectively, and (2)
MPF activation in fish oocytes occurs when preexisting
p34ed2 forms a complex with newly synthesized cyclin B, a
situation differing from that in Xenopus and starfish, in which
the p34¢d:2-cyclin B complex is already present in immature
oocytes.
MOLECULAR MECHANISMS OF ACTIVATION
OF MPF.
M. Yamashita, H. Kajiura, T. Hirai* and Y. Nagahama.
Lab. of Reprod. Biol., Natl. Inst. for Basic Biology,
Okazaki and “Dept. of Biosci., Nishi-Tokyo Univ.,
Yamanashi.
Maturation-promoting factor (MPF) consists of
catalytic p34°4? and regulatory cyclin B. p34°4 is
activated after binding to cyclin B, and forms active
MPF. Using a cell-free system derived from immature
goldfish oocytes, we are investigating the molecular
mechanisms of activation of MPF (p34°**), Activation
of MPF was induced by introduction of cyclin B into
immature oocyte extracts, in which p34°%? was present
but cyclin B was absent. The activation was associated
with phosphorylation of p34°%? on threonine and of
cyclin B on serine. MPF activation was not induced
when phosphorylation was inhibited by protein kinase
inhibitors. To examine whether serine phosphorylation
of cyclin B is required for MPF activation, we pro-
duced a mutant cyclin B which lacks serine residues
(Ser? and Ser”*) phosphorylated by p34°¢?. The
mutant cyclin B was not phosphorylated during MPF
activation, but it was still able to induce MPF activa-
tion. These results indicate that serine phosphorylation
of cyclin B is catalyzed by activated p34‘ and is not
required for MPF activation. Using a mutant p34°¢,
we are currently investigating whether threonine
phosphorylation of p34°%? is a prerequisite for MPF
activation.
PROTEASOME DURING OOCYTE MATURATION AND EGG
ACTIVATION IN GOLDFISH, CARASSIUS AURATUS
T.Tokumoto, M. Yamashita and Y.Nagahama, Lab. of Reprod.
Biol., Natl. Inst. for Basic Biol., Okazaki 444
Changes in protein levels and enzyme activity of proteasome
were examined during 17a,208-DP-induced oocyte
maturation and egg activation in goldfish. Protein levels were
assessed using three kinds of monoclonal antibodies ( GC4/S,
3a and 3B ) against proteasome purified from goldfish
oocytes, each of which is specific for one of the 10
proteasome subunits separated by SDS-PAGE. Enzyme
activity was determined using a fluorogenic peptide ( Suc-
Leu-Leu-Val-Tyr-MCA ) as a substrate. During oocyte
maturation, there were two parallel peaks in the protein levels
and enzyme activity of proteasome, one prior to migration of
the germinal vesicle ( GV ) and the other just after the
completion of GV breakdown. Immediately after egg
activation, a two-fold increase in enzyme activity was
observed, followed by a sharp drop after cyclin degradation.
This decrease in enzyme activity was accompanied by a
decrease in protein levels, except for one minor anti-GC4/S-
positive component which remained unchanged during egg
activation. These results suggest that proteasome is involved
in oocyte maturation and egg activation, and plays a role in
cyclin destruction that occurs soon after egg activation.
GONADOTROPIN INDUCES AN INCREASE IN 17a,20B-
DP RECEPTOR CONCENTRATION AND _ THE
DEVELOPMENT OF OOCYTE MATURATIONAL
COMPETENCE IN A_ TELEOST, THE HIRAME,
Paralichthys olivaceus.
M. Yoshikuni!, Y. Nagahama!, S. Adachi?, S. Ijin2 and K.
Yamauchi?.
\Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki,
2Dep. of Biol., Fac. of Fisheries, Hokkaido Univ., Hakodate.
During the breeding season, hirame spawn daily at a
specified time. Oocytes in various stages of development can
be obtained by sacrifying fishes at various times before
ovulation. Oocytes collected at 30 hr before ovulation
undergo final maturation in vitro in response to human
chorionic gonadotropin (HCG) or 170,20B-dihydroxy-4-
pregnen-3-one (170,20B-DP, a presumptive maturation-
inducing hormone in this species). In contrast, oocytes
collected at 36 hr before ovulation mature with HCG, but not
with 170,20B-DP; however, these oocytes become sensitive to
17a,20B-DP stimulated if exposed to HCG in vitro for 10 hrs.
(3H]17a,20B-DP-binding activity was examined using oocyte
cortices prepared from defolliculated oocytes. Cortices
isolated from 17a@,20B-DP-sensitive oocytes (30 hr before
ovulation) specifically bound [3H]17a,20B-DP, but those from
17a,20B-DP-insensitive oocytes (36 hr) did not. Treatment of
the latter oocytes (36 hr) with HCG in vitro resulted in the
appearance of 17@,20B-DP binding activity. These results
strongly suggest that gonadotropin-induced maturational
competence in hirame oocytes in mediated by an increase in
17a,20B-DP receptor concentrations on the oocyte surface.
Developmental Biology 1163
ARTIFICIAL INDUCTION OF MEIOSIS-RESUMPTION
WITH EMETINE IN PELECYPOD OOCYTES.
K. OSanai and R. Deguchi. Marine Biological
Station, Tohoku Univ., Asamushi, Aomori.
A protein synthesis inhibitor, emetine
is known to induce parthenogenetically
meiosis in metaphase I-arrested pelecypod
oocytes (Mytilus edulis, Dubé and Defresne
1990). We examined whether protein synthe-
sis inhibitors induce meiosis-~resumption
also in prophase arrested oocytes of pele-
eypods (Crassostrea gigas and Limaria ha-
kodatensis). In sea water containing 0. 1-
0.5 mM emetine, the oocytes underwent ger-
minal vesicle breakdown and extruded the
first polar body. After the first meiotic
division, chromosomes’ remained in the
proper oocytes duplicated in number and
aggregated to several clusters, or dispers-—
ed in cytoplasm. The aggregated chromosome
elusters decondensed to swollen nuclei.
The first cleavage often proceeded without
nuclear division. Puromycin (0. 1-0.5 mM)
induced also germinal vesicle breakdown,
but the oocytes were arrested at the first
metaphase of meiosis. These findings
suggest that the mechanism of meiosis
resumption in prophase-arrested oocytes
differs from that in metaphase-arrested
oocytes.
OVARY EXTRACT OF THE ASCIDIAN, HALOCYNTHIA RORETZI1,
THAT INDUCES THE OOCYTE MATURATION AND THE SELF-
STERILITY. 11. Numakunai, and 7H. Yokosawa, Mar.
Biol. Stat., Fac. of Sci., Tohoku Univ., Asamushi,
2Dept. of Biochem., Fac. of Pharmaceutical Sci.,
Hokkaido Univ., Sapporo.
Ovarian egg mass was taken out of the gonad of H.
roretzi by pipetting and suspended in sea water
(sea water:egg mass=1:1). After stirring briefly,
the suspension was centrifuged at 250xg for 15'.
The resulting supernatant was boiled and centri-
fuged at 25,00xg for 30'(ovary extract). The ex-
tract was subjected to a gel filtration Sephadex G-
75 column, and the inhibiting or inducing activity
of the oocyte maturation and the expansion of the
peri-vitelline space(self-sterility) of the ovarian
egg was assayed. When the extract was treated with
0.01% trypsin or allowed to keep O°C for several
days, the inducing activity was detected as two
peaks: the two activities showed different suscep-
tibility to DFP, a serine protease inhibitor. At
the beginning of the spawning season the strong in-
hibitory activity was detected, while near the end
of the spawning season the inhibitory activity was
diminished and inversely the inducing activity be-
came detectable.
The extract of the gonad of Ciona savignyi was
prepared and treated with trypsin. After applying
the Sephadex G-75 column, each fraction was assayed
for the inducing activity using the egg of H. rore-
tzi. The same results were obtained as those in the
extract of H. roretzi prepared near the end of the
Spawning season.
FUSION OF STARFISH OOCYTES WITH DIFFERENT
MATURATION PHASES.
Mitsuki Yoneda. Dept of Zoology, Kyoto
Univ. Kyoto 606
Immature oocytes of Asterina pectinifera
were fused by treating them with poly-
ethylene glycol following the procedure of
Vassetzky et al (1986). Electric pulses
were occasionally applied. 1-methyl-
adenine caused the fused pair to extrude
pairs of polar bodies (PB1's and PB2's) on
schedule of unfused controls, and the pair
eventually formed two pronuclei. Two
populations of oocytes with different
maturation phases were then fused at
random. I observed those fused pairs that
extruded PB2 from one meiotic nucleus
within + 8 min of the PB1 extrusion from
the other nucleus. In these hetero-
geneous pairs, the PB2 was not formed from
the latter nucleus, and both nuclei went
through to form pronuclei. Presence of a
cytoplasmic factor to stop cell division
in maturing starfish oocytes is suggested.
Vassetzky et al (1986) Methods in Cell
Biology, 27:359-378.
REGULATORY REGION OF SEA URCHIN ARYL-
SULFATASE GENE RESPONSIBLE FOR SPATIAL
EXPRESSION.
K.Akasaka, K.Yamada, S.Tanaka*, Y.Iuchi,
K, Takata and H. Shimada. Dept. of Biol.,
Fac. of Sci., Hiroshima Univ., Higashi-
Hiroshima, *Inst. of Life Sci.,
Mitsubishi-Kasei, Machida.
The expression of arylsulfatase(Ars) gene
begins at blastula stage and is restricted
in aboral ectoderm cells in sea urchin
embryos (H. pulcherrimus). We have
demonstrated that the cis-regulatory
elements responsible tor temporal
expression locate in the fragment spanning
from -3kb to -2b of Ars gene by in vivo
transcription assay. In the present
study, we have monitored spatial
expression of CAT(chloramphenicol
acetyltransferase) reporter gene in the
microinjection mediated Ars(-3kb to -2b)-
CAT fusion gene transfered embryos by
using anti-CAT antibody. The expression of
CAT was restricted in the aboral ectoderm
cells as that of endogenous Ars gene and
no ectopic expression was detected. It is
suggested that the cis-regulatory elements
responsible for aboral ectoderm specific
expression also locate in the fragment
spanning from -3kb to -2b of Ars gene.
1164 Developmental Biology
QUANTATIVE ANALYSIS OF THE CIS-ACTING
ELEMENTS REGULATING THE EXPRESSION OF
ARYLSULFATASE (ARS) GENE IN SEA URCHIN
EMBRYO.
K. Yamada, S.Eguchi, K.Akasaka and H. Shima-
da, Zool, Inst, Fac, Sci, Hiroshima Universi-
ty, Higashihiroshima 724, Japan.
We have previously reported that a
pArs-CAT fusion construct, containing the
5’ flanking region of sea urchin (Hemicen-
trotus pulcherrimus) Ars gene spanning
from -2 to -3160, was expressed under a
proper temporal control following injec-
tion into unfertilized eggs. A number of
cis-acting elements regulating the expres-—
sion of the Ars gene was also found by
deletion analysis. As a preliminary step
for quantative analysis of the cis-acting
sequences of the Ars gene, in this report
we describe the factors that affect the
level of Ars gene expression. The results
of DNA titration showed that it is essen-
tial to normalize the CAT reporter activ-
ity to the plasmid contents per embryo is
required for significant quantitation of
the expression level of injected con-
structs. Comparison of the normalized CAT
activity among egg batches suggests that
an experiment using the eggs from a single
batch is required for precise evaluation
of the transcriptional activity of inject-
ed constructs. Quantative analysis of the
upstream enhancer of sea urchin Ars gene
indicates that at least three positive
elements exist in this region.
NEWLY DETECTED CIS-ACTING ELEMENTS
REQUIRED FOR INCREASED EXPRESSION OF THE
ARYLSULFATASE(Ars) GENE THE SEA URCHIN
(H. pulcherrimus) EMBRYO
Y.Iuchi, K.Yamada, K.Akasaka, and
He ShilmadakyeZool einisit-1,.eh aCe SCllan
Hiroshima Univ., Higashi-Hiroshima 724.
When the Ars-CAT fusion gene containing
the Ars sequence spanning from -3000bp to
-2bp was constructed and CAT assayed, the
expression of the reporter gene was fairly
low as compared with that of the
endogenous Ars gene, suggesting the
presence of other cis-acting elements
beyond this Ars sequence.
In the present study, We screened for
new cis-acting elements of the Ars gene
using a microinjection-mediated gene
transfer system, and we found two Ars
sequences which activate the transcription
of the reporter gene. One is in the region
spanning from a part of the lst intron to
the beginning of the 2nd exon, and the
other spanning from the 3rd exon to the
3rd intron. Two distinct DNA binding
proteins that sequence-specifically
interact with these cis-element were
detected by gel mobility shift assays.
SINGLE AND TRIPLE STRAND STRUCTURES
(H-DNA) IN REGULATORY REGION OF
ARYLSULFATASE GENE OF SEA URCHIN EMBRYO.
N.Sakamoto, T. Yamamoto, K. Yamada,
K.Akasaka and H.Shimada. Zool. Inst., Fac.
Sci., Hiroshima Univ., Higashi- Hiroshima
724
We have reported that the upstream
region from —3000bp to -2bp of
arylsulfatase(Ars) gene of sea urchin, H.
pulcherrimus, is responsible for temporal
and spatial regulation of expression of
this gene. This region contains a long
polypyrimidine = polypurine (pyr-pur)
sequence. On the basis of Sil nuclease
sensitivity, we have demonstrated that the
pyr-pur sequence can adopt H-DNA structure
under a moderately acidic condition.
In the present study we have shown that
the pyr-pur region can adopts the H-DNA
configuration even under physiological pH
using DEPC and Os04 as chemical probes.
Cleavage sites of the plasmid containing
the pyr-pur region by the chemical probes
were similar to Sl nuclease reactive sites
under the level of negative supercoiling
higher than those of plasmids isolated
from E. coli.
Since chromosomal DNA is believed to be
under high torsional stresses, it is
likely that the pyr-pur region often form
H-DNA configurations in chromosomal
structures. It is also conceivable that
such an unorthodox DNA structure may have
an important role for regulation of gene
expression.
THE EGIP-BINDING PROTEIN IN EMBRYOS OF
SEA URCHIN, ANTHOCIDARIS CRASSISPINA.
Y.Fujita, K.Yamasu, T.Suyemitsu, and
K.Ishihara. Dept. of Reg. Biol.,
Fac. of Sci., Saitama Univ., Urawa.
We previously reported that sea urchin
embryos contain exogastrula-inducing pep-
tides (EGIPs) throughout the early devel-
opment. In the present study, we investi-
gated the location of EGIP-binding protein
in the embryos.
EGIP-D was incubated with homogenates of
embryos at various stages, crosslinked to
the binding protein using disuccinimidyl
suberate (DSS), and analysed by western
blot using anti-EGIP-D-antibody. As a re-
sult, 30KD protein was detected in both
eggs and embryos.
To examine the localization of this pro-
tein in the surface of embryos, EGIP-D was
added to intact embryos, crosslinked by
DSS, and analysed by western blot. The
EGIP binding protein was detected in, 3nz
tact embryos, but not detected in Ca, Mg-
free sea water (CMF-SW)-treated embryos.
It suggests that this protein exists in
outer surface of embryos and can be
extracted by CMF-SW.
Actually, the effect of EGIP was markedly
decreased on induction of exogastrula for
CMF-SW-treated embryos. The role of EGIP
and its binding protein in surface of
embryos during normal developement are
now under investigation.
Developmental Biology 1165
STRUCTURAL ANALYSIS OF EXOGASTRULA-
INDUCING PEPTIDE GENE
Y.Haruguchi, K.Yamasu, T.Suyemitsu and
K.Ishihara. Dept. of Regulation Biol.,
Fac. of Sci., Saitama Univ.,Urawa.
We previously isolated the cDNA clone
for exogastrula-inducing peptide(EGIP)
precursor of sea _ urchin, Anthocidaris
crassispina and showed that the EGIP gene
is expressed in a stage-specific manner.
In order to elucidate the regulatory
mechanism of EGIP gene expression, we
have tried to isolate genomic clones.
Bulk of the EGIP gene has already been
cloned, but the clones for the transcrip-
tional initiation site and the upstream
region have not been obtained. To com-
plete the cloning of EGIP gene, we plated
the genomic library of sea urchin on
E.coli CES201 strain as a host, and
screened it using 5'-terminal fragment of
EGIP cDNA as a probe. Positive clones
were subcloned into pUC19 vector and
analyzed by Southern hybridization and
sequencing. EGIP gene is at least 7kb in
size and composed of 7 exons and 6
introns. Primer extension revealed that
the capping site is about 100 nucleotides
upstream of 5'-end of cDNA, indicating
that the full-length mRNA is about 1750b.
This result and the sequence of the
genomic DNA upstream of cDNA sequence al-
lowed us to determine the tentative
transcriptional initiation site and the
promotor region.
ANALYSIS OF cDNA FRAGMENTS FOR PROTEIN
TYROSINE KINASES OBTAINED BY RT-PCR METHOD.
K.Yamasu, M.Sakuma, T.Suyemitsu and K.
Ishihara. Dept.of Regulation Biol., Fac.of
Sci., Saitama Univ., Urawa.
Phosphorylation of tyrosine residues in
various proteins by protein tyrosine
kinases (PTKs) is now considered the es-
sential aspect in signal transduction from
outer environment into cells. It seems to
regulate various cell functions such as
proliferation, differentiation, and cell
motility. This prompted us to examine the
roles of PTKs in the development of sea
urchin embryos. As a first step to this
aim, we tried to clone partial cDNA frag-
ments of PTKs by RT-PCR method. Using to-
tal RNA from the embryos of sea urchin An-
thocidaris crassispina as templates and
primers designed to amplify the cDNA frag-
ment corresponding to the highly conserved
PTK catalytic domain, we obtained PCR
fragments for different types of PTKs in-
cluding cytoplasmic PTKs and receptor-type
PTKs. The PCR fragments were obtained
using RNA of all developmental stages ex-
amined ranging from undifferentiated eggs
to pluteus larvae. Northern analysis
showed multiple bands with different
length of 3-6 kb. mRNA of most of the PTK
genes are present in unfertilized eggs and
plutei. These suggest that PTKs are
necessary in oogenesis stage or cleavage
stage and then after gastrulation when
tissue differentiation is initiated.
IN SITU PROTEIN PHOSPHORYLATION IN A SEA
URCHIN EMBRYO LOADED WITH RADIOACTIVE
PHOSPHATE: ANALYSIS BY 2D-PAGE
S.Takeda and H.Hayash
Sugashima Marine Biological Laboratory, School of
Science, Nagoya University, Toba.
We have studied protein phosphorylation in
detail during early developmental stage of sea urchin
embryos. Proteins were labeled in situ by culturing
embryos in artificial sea water containing [$2P]PO4.
The TCA insoluble fractions of cell lysate were
separated by two-dimensional polyacrylamide gel
electrophoresis and phosphoproteins were detected by
fluorography at -80°C.
During the cleavage stage, the pattern of
labeled proteins did not change substantially; i,e.
most spots were found constantly throughout this
period. However, one protein whose apparent
molecular weight was 43 kilodaltons specifically
phosphorylated in pre-hatching blastura. In the case
of Clypeaster japonicus, labeling of this protein was
first appeared at 6.5 hours after fertilization, almost
coincident with 128 cell stage, and intensity of
labeling of this protein was increased until beginning
of hatching. In other species, Anthocidars crassispina
Peronella_japonica_and Mespilia gulobulus, the similar
proteins identically positioned on autoradiograms and
phosphorylated in the similar manner were found.
We have also noticed that the treatment of
embryo with LiCl which is known as vegetalizing
agent caused reduction of the labeling of this
protein. These results suggest that phosphorylation of
43K protein in pre-hatching blastura might play an
important role in embryogenesis of sea urchin
embryo.
PROTEINS TO BE PHOSPHORYLATED IN THE
REACTIONS CATALYZED BY CAM KINASE, C KINASE,
A KINASE AND G KINASE IN SEA URCHIN EMBRYOS.
M.Okuyama, Y.Kamata, S.Furuya and
I.Yasumasu. Dept. of Biol., Sch. of Educ.,
Waseda Univ., Tokyo.
SDS-PAGE of proteins in whole homogenate
of sea urchin embryos was performed after
the exposure of whole homogenate to (f{-*?P)-
ATP in the presence of protein kinase
activators(CAMP, cGMP, Ca**-calmodulin, or
the mixture of Case, TPA and
phosphatizylserine), and protein kinase
inhibitors(HA1004, H-7, H-8, W-5, W-7). On
autoradiograph of SDS-PAGE, protein band
with molecular weight of 63kD was found to
become dense in whole homogenate exposed to
({-*7P)-ATP in the presence of Ca’*-calmodulin
and was faint, when W-7 was supplimented. In
the presence of W-5 at concentrations higher
by about 10 holds then that of W-7, the band
of 63kD protein, to be made dense by Ca**-
calmodulin became quite faint. Dense band of
63 kD protein obtained in the presence of
Ca**-calmodulin was not made faint by H-7 and
H-8. This 63kD protein is probably main one
to be phosphorylated by CaM kinase. Protein
band with molecular weight of 53kD was made
faint by H-7 and H-8. The density of 53kD
protein band was not altered by W-5, W-7 or
Ca**-calmodulin. These proteins were found in
the whole homogenate of morulae, blastulae
and gastrulae.
1166 Developmental Biology
REGULATION OF EARLY EMBRYONIC HISTONE GENE
EXPRESSION BY Ca** SIGNALS IN EMBRYOS OF THE
SEA URCHIN, Hemicentrotus pulcherrimus.
M.Kettoku’, M.Kanda’, K.Mitsunaga-Nakatsubo’,
A.Fujiwara*’ and I.Yasumasu’ *Dept. of Biol.,
sch. of Educ., Waseda Univ., Tokyo, Dept. of
Radiation Res., The Tokyo Metropol. Inst. of
Med. Sci., Tokyo.
It has been reported that pulse treat-
ment with A23187 inhibits hatching enzyme
synthesis but augments *“C-leucine incorpo-
ration into proteins in sea urchin embryos.
The pulse treatment with A23187 for lhr
in pre-hatching period enhanced the rate of
[U-**C]lysine incorporation into H,SO,-
extractable proteins. The enhanced rate of
[U-“*C]lysine incorporation was made low by
tetracaine,ruthenium red and W7, but was
hardly altered by H7 and H8. mRNAs encoding
early embryonic histones were investigated
by Northern blot analysis using genomic DNA
clones of histones Hl and H3 as the probes.
Pulse treatment with A23187 enhanced the
levels of mRNAs encoding histones. The in-
crease in the levels of these mRNAS was also
blocked by tetracaine, ruthenium red and W7
but was not affected by H7 and H8. Probably,
Ca** calmodulin-dependent reactions, enhanced
by A23187-induced artificial Ca’ signals,
enhance expression of several genes such as
early embryonic histone genes in pre-
hatching embryos of sea urchin, though
expression of other genes, such as hatching
enzyme gene, may be turned off by the
signals.
[ADP-RIBOSYL]ATION OF HISTONES IN NUCLEI
ISOLATED FROM SEA URCHIN EMBRYOS
S.Furuya, Y.Kamata, M.Okuyama and
I.Yasumasu. Dept. of Biol., Sch. of Educ.,
Waseda Univ., Tokyo.
Isolated nuclei were exposed to **P-NAD and
histones were extracted by 0O.4N HCl. They
were separated using two-dimensional
electrophoresis of acid/urea/Triton and SDS
gel. Dried gels were analysed for
radioactive proteins by fluorography. These
fluorographs established some differences in
the modification pattern among histones in
morulae and gastrulae. In morulae and
gastrulae, histone Hl is present in mono
modified form and histone H4, at least 4
[ADP-ribosyl]Jated forms. In gastrulae,
histone 13 A= established 11[ADP-
ribosylJated forms and in morulae 10 [ADP-
ribosyljated forms. Histone H3.2 was not
present in [ADP-ribosyl]ated form in
gastrulae but it displayed up to 4 [ADP-
ribosyljated bands in morulae. we AS
expected that mono and poly [ADP-
ribosyl]ated forms of histones have
different functions in nuclei respectively.
Changes in the [ADP-ribosyl]aion pattern
among varients of histones in nuclei of sea
urchin embryo during early development
probably causes alternation of their
functions.
DOES CADP-RIBOSYLJATION OF PROTEINS IN
NUCLEI CONTRIBUTE TO ECTODERMAL CELL
DIFFERENTIATION IN SEA URCHIN EMBRYOS ?
Y.Kamata, A.Fujiwara, S.Furuya and
I.Yasumasu. Dept. of Biol., Sch. of Educ.,
Waseda Univ., Tokyo.
The increase in the ADP-
ribosyltransferase activity followed by its
decrease occurred in pre- and post-hatching
period of early development, with peaks of
the activity at the morula and the gastrula
stage. In embryo wall cells (ectoderm
cells) isolated from sea urchin embryos at
the late gastrula stage, the ADP-
ribosyltransferase activity was markedly
higher than in endoderm and mesenchyme
cells. The increase in the enzyme activity
between mesenchyme blastula and the
gastrula stage, which was blocked by
actinomycin D and cycloheximide, probably
results from the increase in this enzyme
activity in ectoderm cells. Expression of
this enzyme gene is probably augmented in
ectoderm cells. 3-Aminobenzamide (3-ABA),
Evil iy EOS — eye ADP-ribosy!transferase,
inhibited formation of ectoderm structure
more strongly than that of archenterons
(endoderm structure). In animalized
embryos, which exhibited higher activity of
this enzyme than in normal ones, formation
of ectodermal structure was also blocked by
3-ABA. ADP-ribosyltransferase, enhanced in
its activity due to expression of this
enzyme gene in ectoderm cells during
gastrulation, probably contributes to
differentiation of ectoderm cells.
PARTIAL PURIFICATION AND CHARACTERIZATION
OF A CASEIN-KINASE 2-TYPE PROTEIN KINASE
FROM SEA URCHIN
M.Kawamoto?, K.Miyagawa?, T.Natsume? and
I.Yasumasu*. +*Dept. of Biol.,) “Schueeod
Educ., Waseda Univ., Tokyo., *Teikokuzouki
co., LTD., Kanagawa.
It has been reported that the activity of
cyclic nucleotides independent protein
Kinase, is inhibited by heparin in nucleus
fraction of sea urchin embryos. The
activity localized in nuclei is high in
morulae and gastrulae. Extract with 0.4M
NaCl of nucleus fraction isolated from
gastrula was used for the purification of
this enzyme, by sequential chromatography
on Casein-Phosvitin-Sepharose, Heparin-
TOYOPEARL, Sephacry! S-300HR. The finally
obteined enzyme sample exhibited not only
several bands on SDS-PAGE, which were
judged to be subunits of this enzyme, but
also faint bands of other proteins. The
enzyme was only partially purified, though
the specific activity increased markedly
following sequential chromatography.
Partially purified enzyme was’ inhibited by
heparin. The enzyme was relatively
insensitive to spermine, protamine and
poly-:t-lysine and appreciably activated by
poly-,_-arginine.
Developmental Biology 1167
NEW HISTONE MOLECULES PRODUCED AT THE
BLASTULA STAGE OF STARFISH EMBRYOS
T.Shimizu, W.Teramura, T.Tsuruta,
Y.Matsumoto, S.Ikegami. Dept. of Applied
Biochem., Hiroshima Univ., Higashi-
hiroshima, Hiroshima.
We found that the application of
trichostatin A (0.01-100mg/1) to
fertilized eggs of starfish Asterina
pectinifera prevents formation of
msenchymal cells but not cleavages,
blastulation nor gastrulation to occur.
The period sensitive to trichostatin A was
restricted to the midblastula stage (12-14
Iie eto] a8 SNe eal Patra heal(eyey) 4 By using a
monoclonal antibody raised against p29, a
chromatin protein produced during the
blastula stage, we analyzed trichostatin
A-treated chromatins of midblastulae. It
was found that the appearance of p28, a
chromatin protein reacted with anti-p29
antibody, was selectively suppressed by
trichostatin A whereas p29 was present in
the chromatin. Peptide mapping and partial
sequencing experiments revealed that both
p29 and p28 contained an undecapeptide and
an octapeptide. The latter is a part of
the sequence of histone H2B. On the other
hand, the former LS MONE related to
histones, suggesting that p29 and p28 are
new histone H2B anchoring hither-to-
unknown peptide.
PROBABLE PARTICIPATION OF DNA METHYLATION
IN ECTODERM CELL DIFFERENTIATION IN SEA
URCHIN EMBRYOS.
M.Nakasone, Y.Kamata, A.Fujiwara and
Y.Yasumasu. Dept. of Biol., Sch. of Educ.,
Waseda Univ., Tokyo.
In post-hatching period in early
development, evident increase in the rate
of DNA methylation occurred in sea urchin
embryos between the mesenchyme blastula and
the gastrula stage. The rate of DNA
methylation was estimated by the
radioactivity in DNA in nucleus fraction
exposed to [{methy1-14c}s-adenosy1-L-
methionine or in embryos kept with [methyl-
4c¢]methionine. In embryo wall cell
fraction, the rate of DNA methylation was
higher than in archenteron cells and
mesenchyme cells. In animalized embryos,
the rate of DNA methylation was evidently
higher than in the control ones. Exposure
of animalized and normal embryos to SIBA,
which inhibited DNA methylation, strongly
blocked formation of ectoderm structure.
Augmentation of DNA methylation by exposure
to methionine at concentration above im,
which augmented the rate of methylation,
produced abnormal embryos which were alike
to animalized embryos. SIBA canceled the
effect of methionine on embryonic
development. Probably, DNA methylation
contributes to differentiation of ectoderm
cells.
ABNORMAL EMBRYOS DERIVED FROM EGGS TREATED
WITH SCN-.
A. FUJIWARA, T. NAKAGAWA AND I. YASUMASU
Dept. of Biol. School of Education Waseda
Univ., Tokyo.
Sea urchin eggs, kept for 2 hr in arti-
ficial sea water (ASW) containing 0.1-0.3M
SCN- in place of Cis; developed to
animalized embryos. Embryos washed 4
times with SCN--containing ASW during 2 hr
culture with this ASW developed to several
types of abnormal embryos such as
animalized ones and those alike to vegeta-
lized ones. Non-dialyzable compounds
extracted from eggs with SCN- were found
to bind with embryos and exerted animaliz-
ing effect on sea urchin development.
Without washing eggs with SCN--containing
ASW, these compounds, which are extracted
during SCN- treatment, seem to bind again
with embryos upon dilution of SCN-
concentration at the end of the treatment.
Translocation of these compounds is
assumed to result in production of
animalized embryos. Animalizing effect of
these non-dialyzable compounds was
canceled by tetracaine, ruthenium red and
W7. Treatment of embryos with W7, as well
as tetracaine, produced vegetalized
embryos. Ca’, calmodulin dependent
reactions seems to be activated by these
SCN--extractable compounds resulting in
production of animalized embryos.
EFFECTS OF NON-DIALYZABLE COMPOUNDS
EXTRACTED BY SCN- FROM SEA URCHIN EGGS ON
MORPHOGENESIS IN EMBRYOS.
T. NAKAGAWA, A. FUJIWARA AND I. YASUMASU
Dept. of Biol. School of Education, Waseda
Univ., Tokyo
Non-dialyzable compounds extracted with
0.1-0.3M SOM iieroin sea urchin eggs
produced abnormal embryos which were alike
in their morphological characteristics to
animalized embryos. SCN--extract hardly
contained DNA, RNA and substances to be
dissolved with ethanol-ether. This SCN—
extract did not exhibit any activities of
cytochrome c¢ oxidase, rotenone insensitive
NADH cytochrome c reductase, phosphorylase
and glucose-6-phosphate dehydrogenase.
SCN--extract from plasma membrane fraction
isolated from sea urchin eggs exhibited
almost the same activity to cause abnormal
embryo formation to the activity in the
extract from egg homogenate. These
compounds were bound again with embryos.
Thus, we assumed that these compounds is
localized to egg surface. Chromatography
of SCN--extractable matter on Sephadex G-
75 or G-100 column was performed. The
fractions thus obtained, exhibiting a
dense band at the molecular weight of
about 40K as well as other faint bands on
SDS-PAGE, showed the activity to induce
abnormal embryos.
1168 Developmental Biology
INSULIN RECEPTOR OF THE CULTURED CELLS
DERIVED FROM MICROMERES OF SEA URCHIN
EMBRYOS
S.Kuno, T.Nagura and I.Yasumasu.
Dept. of Biol., Sch. of Educ., Waseda
University., Tokyo.
In a previous study, we found that the
micromere-derived cells cultured with
insulin undergo outgrowth of pseudopodial
cables without formation of spicule rods.
Hence, it is assumed that receptors for
insulin or insulin-like compounds exist on
the surface of the micromere-derivrd
cultured cells. In the present study, SDS-
PAGE of proteins in micromere-derived cells
in culture cross-linked with 257-1abeled
insulin was performed to find out receptors
for insulin ( or insulin-like protein ).
Radioautograghs of SDS-PAGE show a band at
the molecular weight of 44k in non-reduced
condition and 41k in reduced condition.
Insulin receptor is known to have protein
tyrosin kinase domein in mammalian cells,
probably, these cultured cells have protein
tyrosin kinase domein. We carried out
immunoblotting with anti-phosphotyrosine
antibody, PY20, to find out the
phosphorylated protein by the protein
tyrosin kinase domein. Immunoblotting
shows bands at the molecular weight of 35
and 55k.
DIFFERENTIATION OF SMOOTH MUSCLE CELLS
DURING HISTOGENESIS OF VESSEL WALL IN
THORACIC AORTA OF MOUSE EMBRYO.
Y. Takahashi. Vessel Research Laboratory,
Fac. of Pharm. Sci., Teikyo Univ., Sagami-
ko, Kanagawa.
As a basis for studying mechanism of
histogenesis of arterial wall, differenti-
ation of smooth muscle cells in thoracic
dorsal aorta of mouse embryo was investi-
gated immunohistochemically using mono-
clonal antibodies against two molecular
markers, a-actin and smooth muscle-
specific myosin heavy chain.
At 9 day of gestation, the aorta con-
sists of endothelial monolayer. Alpha-
actin positive cells appear at 10 day,
when they surround the endothelium as a
single layer. In 11 day embryos, two or
three layers of cells around the endothe-
lium express both a-actin and smooth
muscle myosin heavy chain. At 13 day, 4-5
layers of cells with both markers are
histologically distinguishable from sur-
rounding mesenchyme. The number of smooth
muscle cell layers hardly increases until
15 day of gestation and neonatal period.
In neonatal aortas, there are 1-2 layers
of a-actin negative cells outside the
smooth muscle layers, showing formation of
adventitia.
These results suggest that the initial
phase of possible smooth muscle induction
in mouse aorta occurs before 10 day of
gestation.
CHARACTERIZATION OF MUSCLE-CELL-SPECIFIC
NUCLEAR PROTEINS, P32 AND P30
D. Takai, M. Kirinoki and T. Hirabayashi.
Inst. Biol. Sci., Univ. Tsukuba, Tsukuba.
Taking advantage of the autoimmunity of
some nuclear proteins, we detected and
characterized muscle-cell-specific nuclear
proteins. We injected the protein
components of the nucleus isolated from
chicken muscles into chicken breast muscle.
Three proteins were detected on a 2D-PAGE
pattern of isolated nuclei with the
antiserum. Two of them were not detected in
the nuclei of liver, brain, cardiac muscle
and slow skeletal muscle, but detected in
the nuclei of smooth muscle(gizzard) and
fast skeletal muscle at all stages
examined. They were detected only in a
DNase treated sample prepared in the
fractionation of muscle cell nuclei,
suggesting that they are proteins which
weakly bind to chromatin.
ANALYSIS OF PROTEINS IN MUSCLE NUCLEAR
FRACTION
M. Kirinoki, D. Takai, and T. Hirabayashi,
Inst. Of (Biiely (Sci. sUnsse of Tsukuba,
Tsukuba.
In order to examine the change of
nuclear protein components during cell
differentiation, we compared protein
constituents of nuclei between several
tissues by 2-dimensional gel
electrophoresis. Among proteins which
seemed to have tissue specificity, we
picked up and analyzed proteins that
formed several spots with a molecular
weight of 108 kDa in a range of
isoelectricpoint from pH 7.2 to pH 7.4 on
a 2-dimensional pattern of chicken breast
muscle nuclear fraction. The antiserum
against 108 kDa proteins showec by
immunoblotting tests that i08 kDa
proteins were in nuclear fractions of
gizzard and heart as well as of breast
muscle, but not of brain nor of liver from
1-day-old chicks. The 108 kDa proteins were
first detected in the breast muscle of the
15-day-old embryo. Their amount increased
until hatching and was maintained for 1
week, then decreased gradually. In
sequential extraction experiments, the 108
kDa proteins were found to be soluble ina
solution of high salt concentration after
NP40 treatment and DNase I digestion.
Developmental Biology 1169
TROPONIN T ISOFORM EXPRESSION AND CHIMERIC
FIBERS IN REGENERATING MUSCLE
Y. Yao and T. Hirabayashi, Inst. of Biol. Sci.,
Univ. of Tsukuba, Tsukuba
Using antibodies against anti-fast troponin
T and anti-slow troponin T, we examined the
regenerating muscle cells obtained by
transplantation of anterior latissimus dorsi
(slow muscle) into pectoralis major (fast
muscle) and found by 2-D SDS-PAGE and
immunoblotting that they expressed slow type
troponin T isoforms. Thus, the pattern of
skeletal muscle troponin T isoform expression
is fixed in cell lineage. Futhermore, the
chimeric muscle fibers composed partly of fast
and partly of slow fibers were found in
regenerating muscle blocks, suggesting that
the fast and slow myoblasts fused during
regeneration.
EXPRESSION OF MYOGENIC FACTORS IN
DENERVATED CHICKEN BREAST MUSCLE: ISOLATION
OF THE CHICKEN MYF5 GENE.
2
0.Saitoh!, A.Fujisawa-Sehara“, Y.Nabeshima
, and M.Periasamy>. lDept. of Mol. and Cel.
Neurobiol., Tokyo Metropol. Inst. for
Neurosci., Fuchu, 2 Div. of Mol. Genet.,
Natl. Inst. of Neurosci., Natl. cent. of
Neurol. and Psychia., Kodaira, 3 Dept. of
Physiol. and Biophys., Univ. of Vermont,
Vermot, USA.
2
We have isolated chicken Myf5 gene in
addition to cDNAs for MyoD1 and myogenin,
and analyzed their mRNA levels during
chicken breast muscle development. We found
that chicken Myf5 and MRF4 genes are
tandemly located on single genomic DNA
fragment, and that chicken Myf5 gene is
organized into at least three exons. The
analyses of expression revealed that
myogenin expression is restricted to in ovo
stages in breast muscle, and is not
detectable in neonatal and adult stages.
On the other hand, Myf5 expression is
detectable until day 7 post-hatching and is
not found in adult muscle, whereas high
levels of MyoD1 and MRF4 are detectable at
all stages. To further understand the
roles of innervation during muscle
Maturation, we analyzed the expression of
the myogenic factors in denervated adult
breast muscle. We found that MyoD1,
myogenin, and MRF4 are induced at high
levels in denervated muscle, whereas no
change occurs in the level of Myf5.
CAN COLLAGEN SYNTHESIS TRIGGER THE
EXPRESSION OF MYOGENIN IN CULTURED MUSCLE
CELLS?
R. Matsuda. Dept. of Biol., Col. of Arts & Sci.,
Univ. of Tokyo, Tokyo.
It has been known that the myogenic
regulatory factor (MyoD family) triggers muscle
cell differentiation. In cultured skeletal muscle
cells, the decrement of serum or fibroblast
growth factor concentration is crucial to
activate MyoD family genes. On the other hand,
the extracellular matrix (ECM) is known to be an
important environmental factor to support
muscle cell differentiation. To study the effect
of ECM formation on the expression of MyoD
family gene in cultured BC3H1 cells, we added
200M of ascorbic acid 2-phosphate (a co-factor
for collagen synthesis) into growth medium and
cultured for 4 days. The cells continued to
proliferate and formed well developed ECMs and
multiple cell layers. Immunofluorescent study
revealed that only the cells on the bottom of the
culture dish expressed myogenin. The result
suggests that the enhanced formation of ECM is
one of the important factors to up-regulate the
expression of MyoD family gene during muscle
development.
Involvement of Protein Tyrosine Kinase in the
Regulation of Myogenin Expression
N. Hashimoto, M. Ogashiwa and S. Iwashita.
Mitsubishi Kasei Inst. Life Sci., Machida, Tokyo 194.
Myogenin is one of the muscle-specific transcription
factors that regulate myogenic differentiation
accompanying growth arrest. We are focusing on the
role of tyrosine phosphorylation in the process of
myogenic differentiation of cultured myoblast C2C12
cells.
Using an affinity-purified anti-myogenin antibody,
we revealed the expression pattern of myogenin
during the course of differentiation of C2C12 cells.
Myogenin'was first detected by immunoblot analysis as
a pair of bands prior to myotube-formation and
expression levels remain high in myotubes. To
examine the role of tyrosine phosphorylation in
myogenesis, the effect of genistein, an inhibitor of
protein tyrosine kinase, was examined. When C2C12
cells were cultured for 2 days in differentiation medium
containing various concentrations of genistein,
myogenin expression was suppressed in a dose-
dependent manner. Furthermore, myotube-formation
was also completely inhibited in the presence of 50 uM
genistein. Taken together, these results indicate that
genistein blocks myogenesis through the suppression
of myogenin expression. The present study suggests
that tyrosine phosphorylation plays a key role in the
regulation of myogenin expression and myogenesis.
1170 Developmental Biology
IMMUNOCYTOCHEMICAL EVIDENCE OF BINDING OF ANTI-
BIOTICS TO THE VITELLINE AND FERTILIZATION ENVELO-—
PES OF CHERRY SALMON BGGS AND THEIR BINDING
GLYCOPROTEINS
S. Kudo! and S. Yazawa*. ‘Depts. of Anat. and
?Tegal Med., Gunma Univ. Sch. of Med., Maebashi
The binding of three antibiotics (gentamicin,
Oleandomycin and chloramphenicol) to the vitelline
(VE) and fertilization envelopes (FE) and their
binding glycoproteins were investigated using
cherry salmon eggs. Purified VEs and FEs were
incubated in isotonic solution (pH 7.0) containing
each of the antibiotics (1 mg/10 ml) for 2 hr at
room temperature. After a thorough wash in the
isotonic solution, immunostaining was performed
using antiserum against each of the antibiotics,
followed by photomicrography and immunoelectron
microscopy. Purified VEs or FES were treated with
enzymes (neuraminidase, B-galactosidase, a-manno-
sidase,N-acetyl-8-D-glucosaminidase, and a-L-
fucosidase) , followed by incubation in the anti-
biotic solution and subsequent immunostaining and
immunocytochemistry as described above. The main
binding sites of the antibiotics were demonstrated
to be the VE or FE outermost layer and cortical
alveolus exudates. Enzyme treatment suggested that
the binding of gentamicin and chloramphenicol to
the VE or FE might be related to sialic acid
residues and that of oleandomycin to fucosyl
residues. An extract from the FEs was analyzed by
SDS-PAGE separation, and proteins in SDS slab gels
were transferred electrophoretically to nitrocel-
lulose membranes for immunoblotting analysis. The
binding of the antibiotics was revealed using their
antisera as several bands showing differences and
common features in both position and reactivity.
MEDAKA OOCYTES ROTATE WITHIN THE OVARIAN
FOLLICLE UNDER in vitro CONDITIONS.
T. Iwamatsu. Dept. Biol., Aichi Univ.
of Educ., Igaya-cho, Kariya 448
In a previous study (Zool. Sci., 9 (3):
589-600, 1992), it has been inferred that
in medaka follicles, oocytes surrounded by
a granulosa cell layer may begin to rotate
within the basement membrane before or
when the oocyte axis is established in the
early stage of oogenesis. It is the pur-
pose of this study to ascertain whether oo-
cytes really rotate within follicle. Iso-
lated medaka follicles were incubated in L-
15 Medium supplemented with 10% fetal bo-
vine serum and 40 ug/ml gentamicin sulfate
for a few hours at about 26°C. During in-
cubation, movement of oocytes under a mi-
croscope was recorded on a video cassette
recorder and analysed. Movement of the fol-
licular surface was marked by carbon parti-
cles which stuck to its surface, and the
movement of intrafollicular oocytes was
traced by dislocation of its attaching and
non-attaching filaments on the chorion. Oo-
cytes exhibited the rotation around the
predetermined animal-vegetal axis, some-
times at a slightly oblique angle to the
axis. The velocity of oocyte rotation which
might depend on movement of granulosa cells
was about 30-50 wum/hr and deviated in oo-
cytes of different stages in the early vi-
tellogenic phase of oogenesis. The present
result indicates that medaka oocytes virtu-
ally rotate within follicle.
FORMATION OF FILAMENTS ON THE CHORION OF
OOCYTES IN THE MEDAKA, ORYZIAS LATIPES.
S.Nakashima and T.Iwamatsu. Dept. of Biol.,
Aichi Univ. of Educ., Kariya.
The formation of attaching and non-at-
taching filaments on the chorion of oocytes
of the medaka, Oryzias latipes and the re-
lationship between the distribution of
granulosa cells in the animal hemisphere
(AH) and vegetal pole area (VPA) and that
of attaching and non-attaching filaments
were studied as a step in the clarification
of the mechanism for the determination of
egg polarity.
The oocyte at stage II which is less
than 100 pp in diameter has an almost smooth
surface with a number of tufts of micropro-
jections. Attaching and non-attaching fila-
ments are first recognized as prominent
structures from the oocyte surface at the
end of stage II as reported by Tsukahara
(1971). Most these structures are formed
where adjacent granulosa cells joined by
desmosomes meet, and electron dense materi-
als are observed on the prominent struc-
tures. After that very thin rudiments of
chorion are formed. In stage IV attaching
and non-attaching filaments become uni-
laterally curved candle-like structures.
Iwamatsu (1992) reported that the in-
creasing rate of the distance between fila-
ments is higher in that of AH. Granulosa
cells of AH are larger and flatter than
those of VPA. The difference in the distri-
bution and the shape of granulosa cells may
relate to the difference in the distribu-
tion of filaments in the AH and VPA.
IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF
VITELLOGENIN IN THE KOKANEE SALMON LIVER
AND OVARY.
H.Ueda! and A.Hara2. 1tToya Lake Stn. for
Environ. Biol., Abuta-gun, and 2Nanae Fish
Cult. Exp. Stn., Kameda-gun, Fac. of
Fish., Hokkadio Univ.
Fish vitellogenin (Vg) has been
considered to split into lipovitellin (E1)
and phosvitin-B component (E2) in oocytes.
However, little is known about Vg
production in liver cells and Vg uptake
into oocytes. This study was carried out
to observe immunoelectron microscopic
localization of Vg, El and E2 in kokanee
salmon (Oncorhynchus nerka) liver and
Ovary using specific antisera against chum
salmon (Q. keta) these three proteins by
means of immunogold technique. In the
liver cells, specific immunoreactions of
anti-Vg, El and E2 sera were similarly
detected in the Golgi apparatus and in
granular inclusions of 100-1500 nm in
size. The double labeling technique using
gold particles of different size revealed
that most of these inclusions contained
both El and E2, but some of these
inclusions possessed either El or E2. In
the oocytes, immunoreactive gold particles
to these three proteins were equally
observed in endocytotic vesicles, yolk
granules and yolk globules. These findings
Suggest the possibility that Vg is
produced in the forms of El and E2 in the
liver cells, secreted into blood, and
absorbed into oocytes.
Developmental Biology 1171
Hustches Puninvcation of H=-Sh, Putative
Precursors of Major Egg Envelope Con-
stituents
K. Murata, I.luchi, K.Yamagami Life Sci.
Inst., Sophia univ. ,Tokyo
Spawning female-specific (SF) sub-
stances, putative precursors of major com-
ponents of egg envelope glycoproteins (ZI-
1,-2,-3), can be classified into two groups
in terms of their immunoreactivity and
molecular weight. One is low molecular
weight SF (L-SF), the precursor of ZI-3 and
the other is high molecular weight SF (H-
SF) which presumably includes those of ZI-
Ip Se
H-SF was partially purified from the as-
cites of the Ez2-treated male fish. Ascites
buffered with 20 mM phosphate buffer (pH
6.8) containing 40 mM EDTA and 0.4 mg/ml
PMSF was fractionated with a Sephacryl
S-200 HR column. The fractions containing
H-SF substances were subjected to anti-Cl
IgG affinity column chromatography (Cl:a
purified protein fraction of the hatching
enzyme digests of medaka egg envelope),
followed by fractionation by HPLC with an
AX-300 column. Determination of purity
and the molecular weight of the obtained H-
SF was carried out by immunoblotting
analysis and SDS-PAGE. H-SF seemed to con-
sist of 3 bands of proteins and they were
eluted in the first peak of the AX-300
DEAE silica column. On 8%-gel SDS-PAGE,
these proteins showed the same relative
mobilities as those of the ZI-1,-2.
IMMUNOLOGICAL CHARACTERIZATION OF THE
VITELLINE ENVELOPE COMPONENTS IN THE NEWT,
CYNOPS PHYRRHOGASTER.
T. Suzuki, T. Adachi and K. Onitake. Dept.
of IOI 6 5 Rac. of Seat. p Yamagata
University, Yamagata.
Using the antibody specific to the uterine
egg envelope(UE), we have shown that in
Cynops phyrrhogaster, vitelline envelope
began to be formed in stage II according
to Dumont (1972), and that the follicle
cells surrounding oocytes and some of
liver cells were stained with anti-UE
antibody immunohistochemically. Erle eS
present study, we examined to characterize
the common antigen(s) recognized in the
liver(L-Ag) . Immunoblotting analysis
revealed that molecular weight(MW) of L-Ag
WiclS ue D)- Indirect immunofluorescent
histochemistry using the antibody against
vitronectin(VN) of whose MW was same as
L-Ag revealed that the most outer layer of
UE was specifically stained, while coelom-
ic egg envelope(CE) was never stained.
These results suggests that vitronectin
which is cell-adhesive glycoprotein may be
one of the components of UE. Futhermore,
it was demonstrated that UE of eggs pass-
ing through UP1l corresponds to pars recta
of anura did not react with anti-VN anti-
body immunohistochemically, but the eggs
Passing through UP2 which is posterior
region of UP1 were stained of its outer
most layers of UE. It appears that the
addition of molecules to CE from oviduct
is necessary for the completion of UE.
IMMUNOHISTOCHEMICAL ANALYSIS OF
POLYFUSOME IN EARLY OOGENESIS OF
DROSOPHILA
Y.lwai, F.Maruo and M.Okada. Inst. Biol. Sci.,
Univ. of Tsukuba, Ibaraki.
We have isolated a monoclonal antibody (MAb),
BN461, which reacts with a structure extended
through the ring canals (RCs, cytoplasmic
bridges) within the early germline cell-cluster. On
the other hand, polyfusome (PF) is previously
described as the structure extending through the
RCs. To test the possibility BN461 MAb
recognizing PF, we made precise observations of
the structure reacted with BN461 MAb in whole
mount ovaries using laser-scanning confocal
fluorescent microscopy. 3-D images of the
structure were reconstructed from serial
fluorescent optical sections by using volume
rendering system. In all stages of cystocyte
divisions, the branching patterns of the structure
corresponded to the previously described
branching pattern of PF. Furthermore we found
that there was asymmetrical distribution of
BN461 Ag between two cells that were two
equivalent candidates for the oocyte. Immunoblot
analysis of the sample from ovaries resolved the
Ag into an acidic protein of MW 220kD.
Identification of protein component of the PF in
this study will facilitate molecular and genetic
analyses of PF function during early oogenesis.
SEARCH FOR A DROSOPHILA PROTEIN
HAVING THE SAME EPITOPE OF THE
TETRAHYMENA 49K PROTEIN
T. Arai, Y. Niki, J. Chiba!, H. Suzuki?
and O. Numata“~. Dept. of Biol., PAE 5
Sci., Ibaraki Univ. Mito. Dept. of
Bigil, SCi, Aime Weeing , Sea, Wray, of
Tokyo, Noda. MAH Oi WilOil, Seite,
Univ. of Tsukuba, Tsukuba.
Tetrahymena 49K protein has dual
function to form a 14nm-filament
involved in activity of the pronuclei
during conjugation and to be citrate
synthase in mitochondria. We found that
there presents a protein having the same
epitope of the 49K protein in Drosophila
Ovaries by the methods of Western
blotting and of immuno-histochemistry.
The Drosophila protein having the same
epitope of the 49K protein is 40 kDa and
distributes in the cytoplasm of
developing egg chambers and of the
epithelial and the peritoneal sheath,
muscular tissues surrounding ovaries.
Because of its molecular weight and
characteristics of staining pattern,
this protein is different from actin and
tubulin, and also would not be one of
other known muscular proteins in
multicellular organisms.
1172 Developmental Biology
EFFECTS OF THE BRL-CONDITIONED MEDIUM ON
MOUSE PRIMORDIAL GERM CELLS IN CULTURE.
E.Kawase~’ Y.Shirayoshi~, K.Hashimoto”,
N.Nakatsuji+. ‘Mammal. Dev. Lab., Natl.
Inst. of Genet., Mishima and “Div. of Dev.
Biol., Meiji Inst. of Health Sci., Odawara.
SF (steel factor), LIF (leukemia inhibi-
tory factor) and bFGF promote proliferation
and/or survival of mouse primordial germ
cells (PGCs); The rate of proliferation in
vitro, however, is still much lower than
that in vivo.
Recently, we found that the Buffalo rat
liver cell-conditioned medium (BRL-CM)
promoted survival of PGCs isolated from
12.5 dpe mouse embryos and cultured for 2
days. In this study, we isolated PGCs from
7.5 dpe embryos as allantois fragments or
early 8 dpe (5 or 6-somite stage) embryos
(allantois-hind gut fragments), and cul-
tured them in media with BRL-CM. Our re-
sults indicated that BRL-CM promoted the
proliferation and survival of PGCs. It is
already known that BRL-CM contains soluble
type of SF (rSF) and LIF. Addition of
purified samples of rSF and LIF, however,
gave much smaller effects than BRL-CM.
Moreover, addition of antibodies against
growth factors (TGFB1, EGF or bFGF) did not
inhibit this BRL-CM effect. Therefore,
other unknown/known factor(s) effective on
PGCs seem to present in BRL-CM.
This work was supported by the Science
and Technology Agency, using the special
Coordinating Funds for Promoting Science
and Technology.
RECONSTITUTION OF SEMINIFEROUS TUBULES IN
VITRO BY DISSOCIATED FETAL TESTICULAR CELLS.
K. Hashimoto, T. Mitani, E. Kawase and
N. Takahashi. Div. of Dev. Biol., Meiji
Inst. of Hlth. Sci., Odawara
Primordial germ cells and gonia could be
promising target cells for genetic manipula
tion. We are trying to develop new methods
of production of transgenic mice by using
these cells. Recently, we have succeeded in
obtaining offspring derived from cultured
oogonia (DGD,34,233,1992). However, we have
no methods for obtaining offspring derived
from manipulated spermatogonia.
Fetal testicular cells dissociated with
trypsin-EDTA were plated on partially gell-
ed Matrigel (Hadley et al.,1990). They pene-
trated into the gell and aggregated depend-
ing on the cell density. At low density,
cells were organized into many small
spheres, whereas at high density they first
formed a cell sheet, which was gradually
transformed into a large irregular reaggre-
gate by 12 hr. At about 5x10° cells/cm?
cells were organized into tubules. After 2d
culture, many cells positive for alkaline
phosphatase could be detected in the recon-
stituted tubules. Kuroda et al.(1989) re-
ported that type A spermatogonia differenti-
ated into spermatids when seminiferous tu-
bules isolated from cryptorchid testes were
transplanted into adult testes. Then, we
are trying to transplant these tubules into
adult testes. This study was supported by
the Special Coordination Funds for Promot-
ing Science and Technology.
THE COOPERATIVE ROLES OF GERM CELLS AND
SOMATIC CELLS IN TESTICULAR TERATOCARCINO-
GENESIS IN RECONSTITUTED TESTES IN MICE
M.Noguchi and T.Sugiyama. Dept. Biol., Fac.
of Sci., Shizuoka Univ., Shizuoka
Testicular teratomas can be induced
from germ cells by grafting 12.5-day male
genital ridges to the adult testes in
129/Sv-ter strain of mice, whereas no tera-
toma can be induced by grafting 14.5-day
fetal testes.
In order to analyze the roles of germ
cells and gonadal somatic cells in testicu-
lar teratocarcinogenesis, we examined the
susceptibility to teratocarcinogenesis of
the testes reconstituted from germ cells
and somatic cells in different stages.
The 12.5- and 14.5-day testes and
9.5-day hindguts were dissociated, and
germ cells were separated from somatic
cells. Then, germ cells and somatic cells
were reaggregated by hanging drop-gyratory
culture. The resultant aggregates were
grafted to the adult testes. It was found
that the seminiferous tubules have been
reconstituted in all combinations; 12.5-day
germ cells-12.5-day somatic cells, 12.5-
14.5, 14.5-12.5, 14.5-14.5)5) 905-455 end
that teratomas have been only induced in
12.5-12.5 aggregates, not in any other
combinations. These results suggest that
the cooperative interaction between germ
cells and somatic cells in 12.5-day testes
plays important roles in testicular
teratocarcinogenesis in 129/Sv-ter strain.
THE ter MUTATION, RESPONSIBLE FOR PRIMORD-
IAL GERM CELL DEFICIENCY IS ALLELIC WITH
NEITHER W._LOCUS NOR S1 LOCUS IN THE MOUSE.
M.Noguchi~, T.Sakurai~* K.Moriwaki and
H.Katoh*. 1bep.of Biol., hac ohescresona
zuoka Univ.,Shizuoka, “Yokohama City Univ.,
Yokohama, ~Natl. Inst.of Genetics, Mishima,
Central Inst.for Exp. Animals, Kawasaki
A recessive mutation, ter(teratoma) cau-
ses primordial germ cell(PGC) deficiency
in ter/ter mice of both 129/Sv-ter strain,
in which ter also causes a high incidence
of congenital testicular teratomas(Noguchi
and Noguchi.1985, Noguchi et ai.1985), and
ter congenic strains, C57BL/6J-ter(Bé6-
ter) and LTXBJ-ter (Noguchi et al. 1987,
1988, Noguchi and Kobayashi. 1991). The
series of W(dominant white spotting, Chr
S) and S$l(Steel, Chr 10) mutations also
cause the PGC deficiency in their homozy-
gotes.
In order to examine the allelism of ter
with W or Sl locus, the linkage tests betw-
een ter and Si) or Pgm-1(phosphoglucomu-
tase-1) which is mapped about 3 cM from
WwW were Carried out, by using offspring
produced by genetic test crosses between
+/ter mice of Bé-ter and DBA/2J, C3H/HeJd
or 129/Sv-S1CP.
The results showed that ter was neither
linked to nor allelic with Sl and WwW
(the vicinity of Pgm-1), indicating genet-
ically that ter is a new mutation which
affects the nature of PGCs, differing from
Wand Sl.
Developmental Biology 1173
SEARCH FOR FACTORS RELATED TO SEX
DIFFERENTIATION IN FISH
Y. Fujiwara! , T. Hirabayashi? and J.-I.
Miyazaki?. 'Inst. of Envi. Sci., ?Inst. of
Biol. Sci., Univ. of Tsukuba, Tsukuba.
A temperate wrasse (Halichoeres
poecilopterus) is known to undergo sex
reversal (protogyny). To detect factors
related to sex differentiation which seem
to appear during sex transition, we
compared protein constituents between
transitional and mature gonads by _ two-
dimensional gel electrophoresis. By
comparison between transitional gonads and
Ovaries, six proteins were found to be
specific to or significantly increased in
the transitional gonads. Three of the six
proteins were found in testes. We prepared
an antiserum against whole ovarian
proteins. After incubating the antiserum
and whole testicular proteins and removing
immuno-precipitates by centrifugation, the
Supernatant was used to prepare an
antiserum against testis-specific proteins.
The antiserum recognized one of the
proteins shared by transitional gonads and
testes. The protein had an apparent
molecular weight of 26 kDa and an
isoelectric point at pH 5.3 and was not
detected in the liver and muscle. The
protein is a possible candidate for the
factor which is related to sex
differentiation and expressed continuously
in mature testes.
HETEROCHRONIC pPGCs IMPLANTED INTO HOST NEURULAE
CAN MIGRATE, TOGETHER WITH HOST pPGCs, TO GENITAL
RIDGES OF EXPERIMENTAL TADPOLES.
T. S. Tanaka and K. Ikenishi. Dept. of Biol., Fac. of
Sci., Osaka City Univ., Osaka.
In normal development of Xenopus, presumptive
primordial germ cells (pPGCs) which have acquired
the migratory activity at around the mid-tailbud
stage are thought to be able to migrate to the
dorsal crest of endoderm by responding a signal
from the dorsal axial mesoderm. Then, they emerge
from the crest through the dorsal mesentery and
migrate to genital ridges at the tadpole stage,
responding to fibronectin. When the labelled pPGCs
in explants at the same 'age' as host pPGCs were
implanted into unlabelled host neurulae, they
could migrate, together with host pPGCs, to the
genital ridges of the experimental tadpoles
(kKenishi et al., 1984). Employing essentially the
same experimental system as in the previous study,
we investigated whether implanted pPGCs of
different ‘age' from host pPGCs can migrate,
together with host pPGCs, to the genital ridges of
the experimental tadpoles in response to the
signal.
There were no _ significant differences in
Proportions of the experimental tadpoles with
labelled PGCs, irrespective of the '‘age' of
implanted pPGCs. This indicates the implanted,
heterochronic pPGCs can migrate to the genital
ridges of the host tadpoles, responding the signal
properly.
STRUCTURES OF CORTICAL ALVEOLAR-DERIVED
GLY COPOLYPROTEINS (HYOSOPHORIN) FROM
THE EGGS OF MEDAKA FISH SPECIES - ORYZIAS
LATIPES (ORL) AND O.MELASTIGMA (ORM)
T. Taguchi’, A. Scko’, K. Kitajima’,
Y. Inoue’, S. Inoue’, and T. Iwamatsu?
Dept. of Biophys. & Biochem., Fac. of Sci., Univ. of
Tokyo, Tokyo, *School of Pharm. Sci., Showa Univ.,
Tokyo, *Dept. of Biol., Aichi Univ. Educ., Kariya.
We determined the complete structure of L—hyosophorins
isolated from two species of medaka fish. Orl and Orm. They
were shown to have the identical core peptide sequence: Asp-
Ala—Ala—Ser—Asn(CHO)-GIn-Thr-Val-Ser. The structures
of their glycan chains (CHO) were found to have some
characteristic features as summarized below: (i) The glycan
units of Orl and Orm hyosophorin share commonality in
having (a) a huge N-linked form of which apparent
molecular weight is ~7K, (6) a novel galactose clusters
(Galf1—+4GalB1—4Galf1—), and (c) uniquely branched
galactose residues (4GIcNAch1—3(—4GalB.1—4)GalB1—);
(ii) L-hyosophorin (Orm) has a tetraantennary glycan unit
while Orl L—hyosophorin contains a pentaantennary unit;
(iii) The sialic acid residues (NeuSAc) are a2,3-linked to the
terminal galactose residues in Orm hyosophorin whereas they
were a2,3—linked to the terminal and/or penultimate galactose
residues in Orl hyosophorin: (iv) Or! hyosophorin contains
fucose residues, but Orm hyosophorin is totally devoid of
them.
DETACHMENT OF THE GLYCAN CHAIN FROM
HYOSOPHORIN DURING EARLY EMBRYOGENESIS OF
MEDAKA
ASekol, K.Kitajima!, Y Inouel, S.Inoue2, IDept. of
Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo, Tokvo.
2School of Pharm. Sci., Univ. of Showa, Tokyo.
We found in early embryos of medaka a free glycan chain
having the structure identical with that of the sugar chain of
hyosophorin, a cortical alveolar glycopolyprotein. The free
glycan, not present in unfertilized eggs, was shown to be
liberated at the stage of gastrulation from L-hyosophorin which
was formed from hyosophorin on fertilization. Either the apo-
peptide or the free glycan derived from L-hyosophorin may
possibly be relevant to biological function of L-hyosophorin.
In this study, we identified, in early embryos of medaka, the
presence of an_N-glycanase responsible for the detachment of
the glycan chain, and characterized the partially purified
enzyme.
1174 Developmental Biology
ISOLATION AND CHARACTERIZATION OF
HYOSOPHORIN FROM BUFO JAPONICUS
Y. Shimoda’, K. Kitajima’, Y. Inoue’ and S. Inouc’.
‘Dept. of Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo,
Tokyo; *School of Pharmaceut. Sci.. Showa University, Tokyo
With the aim to isolate cortical alveolar—derived
carbohydrate rich glycopolyprotein (hyosophorin), we applied
the isolation procedures established for fish eggs to Bufo
japonicus eggs and carried out structural determination of a
glycoprotein thus obtained. In brief, after removal of jelly
from the fertilized eggs of B. japonicus, the homogenate was
treated with phenol. Following dialysis of the aqueous phase,
it was chromatographed on a DEAE-Sephadex A-25 column.
When the fraction eluted at ~0.15 M NaCl was then
subjected to Sephacryl S—200 chromatography, we obtained
a glycoprotein which eluted at position slightly after
breakthrough. Interestingly, this glycoprotein was found to
have amino acid and carbohydrate compositions closely
similar to those of hyosophorin of Oryzias latipes.
Sequence analysis of the core protein of the Bufo
glycoprotein revealed that it comprises tandem repeats of
nonapeptide exactly identical with apo—H—-hyosophorin of O.
latipes. Furthermore, methylation analysis of the huge
carbohydrate chains showed the presence of a number of
partially methylated alditol acctates which mostly share with
those formed upon methylation analysis of the O. latipes
hyosophorin, strongly indicating that the glycan units of the
Bufo hyosophorin has a N-linked bulky pentaantennary
structure. The possible physiological significance of the
present findings of closely similar hyosophorin molecules in
toad and medaka is considered in the light of their unique
structural features.
IMMUNOELECTRON MICROSCOPE OBSERVATIONS ON
THE FORMATION AND DEVELOPMENT OF CORTICAL
GRANULES IN XENOPUS LAEVIS OOCYTES.
N. Yoshizaki, Dept. of Biol., Fac. of Gen.
Educ., Gifu Univ., Gifu.
The origin and development of cortical
granules were observed by treating sections
of Xenopus laevis oocytes with a rabbit
antiserum against cortical granule lectins
and with a gold-conjugated goat antiserum
against rabbit IgG. In stage I oocytes,
gold particles were present on small
numbers of cortical granules of 200-600 nm
size. In stage II and III oocytes, they
appeared on granules in the Golgi complexes
as well as on large numbers of cortical
granules, ranging in size from 200 nm to
1.4 um, in the cortical cytoplasm. Some of
these cortical granules showed an irregular
shape, indicating fusion of small granules
into a large one. Gold-labeled granules
disappeared from the Golgi complexes at
stage IV. Cortical granules in stage V
oocytes consisted exclusively of large
granules and they were alined beneath the
oolemma at stage VI. These results suggest
cortical granules form in the Golgi
complexes of stage I to III oocytes and
coalesce in the cortical cytoplasm of stage
i to" LV oocyvelss.
VITELLIN COAT LYSINS FROM Mytilus edulis
SPERM.
T.Takagil, A. Nakamura?, R. Deguchi? and K.
Kyozuka? 1lBiol. Inst., Fac. Sci., Tohoku
Univ., Sendai, Dept. Pharmacol., Gunma
Univ., Sch. Med., Maebashi, 3Marine Biol.
Station, Fac. Sci., Tohoku Univ., Asamushi
The acrosomal proteins obtained from
Mytilus edulis were separated by a reverse
phase HPLC. They were separated into 11
peaks and 3 of them (M3, M6, M7) showed
strong vitellin coat lysin activity. The
amino acid sequences of these proteins were
determined by peptide sequence analyses.
The sequence of M7 was confirmed by cDNA
sequence analysis and revealed to have a
signal peptide of 38 residues. M6 and M7
were composed of 180 amino acid residues and
sequences were 76 % identical. On the other
hand, M3 was composed of 148 residues and
the sequence was different from M6 or M7.
No sequence homology with lysins of abalone
and Tegula was observed. Although the
sequence of M3 was different from those of
M6 and M7, all three proteins have a typical
C-type lectin structure. No lectin activity
was observed, but they were coprecipitate
with isolated egg membrane. Mytilus lysins,
M3, M6 and M7 are supposed to recognize the
carbohydrate moieties of proteins involved
in vitellin coat membrane and bind them and
destroy the the structure of membrane.
These processes can explain that
stoichiometrical amount of lysin is
necessary to destroy membrane.
SPECIES-SPECIFIC SEQUENCES OF TEGULA
VITELLINE COAT LYSJNS.
K. Hanno SR ase! SONI: Sakai2, M. Tanaka
& Y. Nagahama“. ~Tokyo Metropol. Univ.,
Tokyo, & “Natl. Inst. for BasvesRiaiis
Okazaki.
The vitelline coat lysin (VCL) of Tegula,
a marine Mollusca genus, is released from
acrosomal vesicles of the sperm during
acrosome reaction and can lyze the vitel-
line coats of only the same species. Thus,
the lysin action is extremely species-
specific.
cDNA libraries were constructed from the
testicular poly(A) RNAs of Tegula rustica
(Owase) and T. lischkei with lambda gtl0
phage as a vector. A clone bearing the mRNA
sequence for each VCL was isolated by
screening the corresponding libraries with
a 5’-terminal region; 400 bp (ORF-370) was
obtained from the cDNA clone for VCI. of 7.
pfeifferi. The cDNAs of T. rustica (Owase)
and T. lischkei contained an open reading
frame encoding 162 and 173 amino acid
residues, respectively, and indicated the
occurrence of 22 residues of signal se-
quence at the amino terminal region of the
nascent peplide. Both deduced amino- and
carboxyl-proximal domains were virtually
identical lo those determined for the VCIL
of T. pfeifferi. However, the deduced
sequences at the central domain (position
77-92) were different among the three
species. This variable domain may account
for the species specificity of the lysin.
Developmental Biology 1175
A 70KD GLYCOPROTEIN ISOLATED FROM THE
VITELLINE COAT IN THE ASCIDIAN,HALOCYN-
THIA RORETZI
S. Takizaw and M. Hoshil»2 1 Dept.of
Eusewscie, -Gene Res. Cent, fokyo Inst. of
Tech., Tokyo)
Sugar chains of vitelline coat glycopro-
teins are important for sperm-egg interac-
tion in the ascidian, Halocynthia roretzi.
Since no information is available on the
chemical structure of the protein portion
of the vitelline coat, we have isolated
the major glycoprotein of 70kDa mainly due
to the insolubility of the vitelline coat.
We found that the major glycoprotein of
70kDa was selectively solubilized from the
vitelline coat by DMSO. The solubilized
glycoprotein was purified by SDS-PAGE and
digested by lysylendopeptidase. Two major
fragments purified by SDS-PAGE are under
the process of sequencing.
PROPERTIES OF A GLYCOSIDASE PURIFIED
FROM EGGS OF THE ASCIDIAN,
HALOCYNTHIA RORETZI.
K. Matsuura, H. Sawada, and H. Yokosawa.
Dept. of Biochem., Fac. of Pharmaceutical Sciences,
Hokkaido University, Sapporo.
In ascidians, Lambert has proposed that an
egg glycosidase plays a key role in establishment of
the polyspermy block through its binding to the
sperm receptor on the vitelline coat.
Fertilization of the ascidian, Halocynthia
roretzi, was inhibited by GlcNAc-specific lectin
(WGA) and the GleNAc'ase activity was highest
among glycosidase activities released from the eggs
by Ca2+ ionophore. Standing to the above proposal,
these results imply the involvement of the egg
GleNAc'ase in polyspermy block of H. roretzi. The
GlcNAc'ase was purified from eggs to apparent
homogeneity by chromatographies on DEAE-
Toyopearl, SP-Toyopearl, Sepharose 6B, and Mono
S. The molecular weight of the enzyme was
estimated to be 520K by gel filtration and 66K by
SDS-PAGE, suggesting the oligomeric nature of the
enzyme. Isoelectric point was determined to be 7.0.
The enzyme was activated with the isolated vitelline
coat in artificial seawater. This result suggests that
the GlcNAc'ase secreted from the eggs upon
fertilization is activated in contact with the vitelline
coat and probably functions in the polyspermy block.
EXOCYTOSIS DURING FERTILIZATION OF A SEA
URCHIN EGG DETECTED BY FLUORESCENCE
DEQENNCHING METHOD
Kazuhisa Takemoto’, Ken-ichi Hirano’,
Tsuyoshi Hayakawa® and Hideyo Kuroda’.
‘Sugashima M.B.L., Fac. of Sci., Nagoya
Univ., Toba 517, “Tsukuba Res. Lab.,
Hamamatsu Photonics, Tsukuba 300-26 and
*Biol. Inst., Fac. of Sci., Toyama Univ.,
Toyama _ 930.
In general, it is not easy to observe
directly the exocytosis of living cells
under a light microscope. We attempted to
detect the exocytosis during in the
fertilization of sea urchin eggs under a
microscope using fluorescence dequenching
technique with a membrane probe, 5-(N-
octadecanoyl)aminofluorescein (AF18). The
dye dissolved in the lipids of cell
membranes was self-quenched at the
concentration up to 5 mol%. When an AF18-
stained egg was fertilized, the
fluorescence increase was observed and
propagated from the point of sperm entry to
the opposite for 25 sec. The fluorescence
increase is due to the dequenching caused
by the diffusion of AF18 from plasma
membrane to cortical vesicles during
exocytosis. From the simultaneous
measurement of the changes in fluorescence
and light scattering which reflects
exocytosis, the delay from exocytosis to
dequenching was within 1 sec. These results
show that the fluorescence dequenchching of
AF18 is useful for studying exocytosis.
SURFACE MEMBRANE RETRIEVAL VIA ENDOCYTOSIS
IN THE DARK WAVE OF ACTIVATION WAVE FOLLOW-
ING THE CORTICAL GRANULE EXOCYTOSIS IN THE
EGG OF XENOPUS LAEVIS.
T.Kageyama and H.Kubota. DYE, Cyt Iai < |,
Kyoto Pref. Univ. of Medicine, Taishogun,
Kee a kupmiyiOlCO MOOS manda) e pit mon ZOolin
Fac. of Sci., Kyoto Univ., Kyoto 606.
Unfertilized eggs were dejellied, and
the yolk membranes were removed manually.
Denuded eggs were artificially activated by
pricking. Artificially inseminated eggs
were used for following the later develop-
ment. The internalization of surface
membrane was examined in whole mount prepa-
ration with Lucifer Yellow CH by fluores-
cence microscopy, and ultrastructurely with
horseradish peroxidase (HRP) as the marker
of fluid phase endocytosis. Unfertilized
eggs did not internalize the marker. The
endocytosis began and was most prominent in
the region where the dark wave (0.01-0.05;
normalized time after the fertilization.
1.0; the first cleavage) of activation wave
was passing through, immediately after the
cortical granule exocytosis in the white
wave (0-0.04)(Takeichi & Kubota, 1984).
Elongated microvilli were reshortening to
become globular in the dark wave. Endocyt-
ic activity was detected in the region
around the (presumptive) polar body in the
early half till the first cleavage, and in
the stress folds around the cleavage furrow
during the cleavage stage.
1176 Developmental Biology
BOTULINUM EXOENZYME C3 INDUCES ELEVATION
OF THE VITELLINE COAT OF ASCIDIAN EGGS.
S. Toratani and H. Yokosawa. Dept. of
Biochem., Fac. of Pharmaceutical Sci.,
Hokkaido Univ., Sapporo.
In the fertilization process of the
ascidian, Halocynthia roretzi, expansion
of the perivitelline space, called
elevation of the vitelline coat, is
observed within 30 minutes after
insemination or addition of calcium
ionophore. This phenomenon is thought as
a result of exocytosis of intracellular
granules likely as elevation of the
fertilization membrane of sea urchin eggs.
In this study, we examined the effects
on elevation of the vitelline coat of H.
roretzi eggs of botulinum exoenzyme C3, an
enzyme which transfers ADP-ribosyl moiety
from NAD to low molecular weight (LMW)
GTP-binding proteins, and a monoclonal
antibody which inhibits C3-catalyzed ADP-
ribosylation, using microinjection
technique. Elevation of the vitelline
coat was induced by C3 and the elevation
was inhibited by heparin that was co-
injected with C3. The monoclonal antibody
inhibited elevation induced by
insemination, but its inhibition was
canceled by the subsequent addition of
calcium ionophore A23187. These results
suggest that LMW GTP-binding protein(s)
activated upon fertilization stimulates
mobilization of intracellular calcium,
which induces exocytosis.
THE ABILITY OF AXIAL POLARITY EXPRESSION
IN BODY FRAGMENTS OF POLYANDROCARPA
MISAKIENSIS DURING CHIMERA FORMATION.
T.Ishii and Y.Saito. Shimoda Mar. Res.
Ctr., Univ. of Tsukuba, Shimoda, Shizuoka.
In the compound ascidian, Polyandrocarpa
misakiensis, when two body fragments from
different adult zooids fuse with each
other at their cut surfaces, a chimera
zooid is formed. In the present study, to
examine the ability of axial polarity
expression of each body fragment during
chimera formation, four kinds of fragments
were used: normal anterior fragments (NAF)
and normal posterior ones’ (NPF) from
normal situs viscerum (NSV) zooids, and
reversed anterior ones (RAF) and reversed
posterior ones (RPF) from situs inversus
viscerum (SIV) zooids. Analyzing two axial
polarities, anteroposterior (A-P) one and
heart-gut (H-G) one of obtained chimera
zooids, it was found that there were
differences in the ways of A-P axial
polarity expression and H-G one. The
results showed some differences of
expression ability among the body
fragments of NSV and SIV zooids; The NPF
had the most stable ability in two axial
polarity expressions, because it had
expressed own axial polarities whomever it
fused with. On the other hand, the ability
of the RAF was the weakest in chimera
formation. It could not express own axial
polarities, when it fused with an NAF or
an NPF.
EFFECTS OF X RAY AND/OR MAGNETIC
FIELDS ON SEA URCHIN DEVELOPMENT.
N.Uto!, Y.Yamahama’, M.Takai’, T.Masui¥,
and M.Kaneko*’ '‘Dept.of Biol.,?Dept.of
Radiol., Hamamatsu Univ. School of
Med., Hamamatsu.
it has been reported the biological
action of X ray or electromagnetic
fields on development respectively.
In the present study, we investi-
Gated the effects, specially multiple
effects, of X ray and magnetic fields
on development of sea urchin embryo.
Exposure of X ray (65 cGy/min) for 4-8
min on sea urchin (Hemicentrotus p.)
caused a significant delay on early
development. The influences of static
magnetic field (2 Tesla) for 30 min on
development appear negligible. Wheras
changing magnetic field for 90 min
caused a little number of short-armed
plutei in some cases in spite of no
developmental delay. Some of embryos
exposed to both X ray and magnetic
field tend to be in developmental
abnormalities and delay. Amplified
effects of magnetic field on X lay
were uncertain.
The present study will extend the
information about potential effects on
growing body cell.
THE PARTICIPATION OF PHOSPHORYLATED PRO-
TEINS IN MICROTUBULE NUCLEATING ACTIVITY
OF MTOG ISOLATED FROM SEA URCHIN EGG MITO-
TIC APPARATUS.
E.Ueda! .E.Okumura? ,S.Hisanaga’” ,T.Kishimoto
2 and M.Toriyama!. 'Dept. of Biology, Fac.
of Liberal Arts, Shizuoka University,
Shizuoka and ?Lab. of Cell and Develop-
mental Biology, Fac.of Biological Science,
Tokyo Institute of Technology, Yokohama.
A dramatic change in microtubule net-
work occurs at the transition from inter-
phase to M-phase. This change includes
the increase in microtubule nucleating
activity of centrosomes. To elucidate the
activation mechanism of centrosome at M-
phase, we isolated microtubule organizing
granules (MTOGs) from sea urchin mitotic
apparatus. The activity of MTOG decreased
upon incubation with alkaline phosphatase.
MPF but ATP restored the nucleation capa-
city of dephosphorylated MTOGs. Treatment
with 1M KCl inactivated MTOGs by solubili-
zation of the component (s) responsible for
microtubule nucleation. But sea urchin
egg extract restored the microtubule nuc-
leation. The extract contained proteins
which were phosphorylated by MPF. In the
SDS-PAGE these proteins had the same mobi-
lity as the proteins that was existing in
MTOG fraction and was phosphorylated by
MPF. These results suggest the partici-
pation of phosphoprotein(s) in the acti-
vity of MTOG.
Developmental Biology 1177
DEFORMATION OF THE NUCLEAR ENVELOPE CAUSED
BY MICROTUBULES DURING CELL DIVISION IN
SEA URCHIN EGGS.
Y. Hamaguchi, M. S. Hamaguchi and S. Sato.
Biol. Lab., Fac. of Sci., Tokyo Inst. of
Technol., Tokyo.
In order to investigate the process
of nuclear disappearance, the image of the
nucleus in the living cell was improved
with the image processor, Image-1
(Universal Imaging Corp.). Clypeaster
japonicus eggs were fertilized, deprived
of the fertilization envelope and cultured
in Ca-free sea water. The nucleus was
clearly observed at prophase in the egg,
centrosomes were positioned on the edges
of the nucleus which became football-
shape, and the nuclear envelope was
dimpled near the centrosomes. Just before
the disappearance, projections appeared at
the dimpled areas in the nucleus and
elongated deeply up to 5 um or more into
the nucleus with time. Then the image of
the nucleus became obscure, which means
that the envelope broke down. Those
projections were observed not only at the
first cleavage but also at successive
divisions. In the eggs treated with
colcemid, there was no projection. By
immunofluorescence microtubules were
observed in the nucleus before the
disappearance as well as around the
nucleus. Therefore, the nuclear envelope
mechanically pressed into the nucleus by
microtubules from the centrosomes may be
observed as the projections.
RELATIONSHIP BETWEEN ASTER FORMATION AND
CELL DIVISION THROUGH MATURATION TO
CLEAVAGE REVEALED BY TRANSPLANTATION OF
THE STARFISH CENTROSOME.
T. Saiki and Y. Hamaguchi. Biol. Lab.,
Fac. Sci., Tokyo Inst. Tech., Tokyo.
In order to investigate differences
among the first and second maturation
divisions, and the first cleavage
division, we transplanted a centrosome at
each division into maturating oocytes or
zygotes of the starfish, A. pectinifera.
In the zygote, the centrosome extracted at
every divisions formed an aster, which
induced cytokinesis, and eventually the
recipient zygote divided into three
blastomeres after the first cleavage. The
centrosome extracted at any division also
formed an aster in maturating oocytes.
However, the polar body extrusion always
eccurred normally; namely, an aster
additionally formed in the recipient
eocyte never induced cytokinesis.
Stabilizing the mitotic aster with
hexylene glycol treatment demonstrated
clearly that the size of the aster in
maturation divisions was smaller than that
in the first cleavage. Asters reduced in
size in the first cleavage with colcemid
treatment failed in inducing cytokinesis.
In conclusion, 1. the centrosome has no
functional difference in aster formation
among maturation and cleavage, and,
however, 2. the size of the resultant
aster determines whether or not cell
division is induced.
DETERMINATION OF THE MOTIVE FORCE
GENERATED IN THE CELL USING A CENTRIFUGE-
MICROSCOPE.
Y.Hiramoto and M.Kikuyama. slob, IeWorS 5
Univ. of the Air, Chiba.
We designed and constructed a new
centrifuge-microscope with which
microscopic objects can be observed
through differential interference contrast
optics with a 40x objective lens during
application of a centrifugal force up to
1000x gravity. It consists of a Nikon
inverted microscope (TMD), a custom-made
centrifuge machine, a flash lamp with a
very short flash duration (170ns), and a
video-recording system. The cell ina
centrifuge chamber being spun was
illuminated with a flash whenever it
passed through the front of the objective
lens, and its microscopic image was
recorded with the video-system.
We tried to determine the motive force
for chromosome movement during anaphase of
mitosis by measuring the speeds of
chromosomes in a centrifugal field applied
in the direction of the spindle axis. The
chromosome speeds should increase or
decrease by the forces applied to the
chromosomes as a result of the centrifugal
acceleration and the density difference
between the chromosome and the surrounding
protoplasm. Experiments in sea urchin eggs
and in grasshopper spermatcytes are in
progress. It is expected that force-
velocity relations in chromosome movement
are determined.
BEHAVIOR OF NUCLEI AND CHROMOSOMES FOR
PARTHENOGENETIC STARFISH EGGS TO ESTAB-
LISH TETRAPLOIDY
Setsuko Washitani-Nemoto and Shin-ichi Nemoto
Biol. Lab., Hitotsubashi Univ., Kunitachi, Tokyo and
Tateyama Marine Lab., Ochanomizu Univ., Koh-yatsu
Umi-no-Hoshi, Tateyama, Chiba, Japan.
In starfish, artificial parthenogenesis Is associated with
suppression of polar body (PB) extrusion. Both of par-
thenogenetic eggs lacking PBs (OPB eggs) and eggs
with 1st PB alone (1PB eggs) develop as tetraploids. To
know how and when they become tetraploid, nuclear
behavior of the parthenogenetically stimulated eggs of
Asterina pectinifera (2n=44) was examined by stain-
ing them with aceto-orcein.
OPB eggs: After 22 tetrad chromosomes finally divided
into 88 monads through two rounds of separations, they
came together to form a single nucleus. This then
disappeared and a diaster spindle was formed to divide
88 bivalents into two groups, followed by first cleavage.
1PB eggs: After meiotic 22 dyads divided into monads,
they came together to form a single nucleus. When it
disappeared, only a_half spindle was formed. Forty four
bivalents separated into 88 univalents, but the eggs
failed to cleave. In the next round of mitosis, a diaster
spindle appeared to cleave the eggs into two blasto-
meres, each of which contained 88 chromosomes.
Thus, both types of the parthenogenetic eggs, OPB and
1PB eggs, become tetraploid before first cleavage.
1178 Developmental Biology
DNA REPLICATION FOR PARTHENOGENETIC
STARFISH EGGS TO ESTABLISH TETRAPLOIDY
A. Nomura’ and S. Nemoto2. ‘Dept. of Zool., Fac. of
Sci., Kyoto Univ., Kyoto, Japan, 2Tateyama Marine Lab.,
Ocha-no-mizu Univ., Umi-no-Hoshi, Tateyama, Japan.
In starfish, suppression of polar body (PB) extrusion is
prerequisite for production of parthenogenetic
development. Eggs lacking PBs (OPB eggs ) have
chromosomes equivalent to tetraploid , and eggs
bearing 1st PB only (1PB eggs) have diploid . Despite of
the difference of ploidy, both types of parthenogenetic
eggs develop to tetraploid embryos.
In the present study, we timed the DNA replication
periods (S phase) up to the first cleavage in these
parthenogenetic eggs of Asterina pectinifera. The eggs
were labeled with BrdU and the incorporated BrdU was
detected with anti-BrdU monoclonal antibody.
In the both types of parthenogenetic eggs, each S
phase was detected on the same schedule as would be
observed in normally fertilized eggs. In the 1pb eggs,
they did not cleave between first and second S phase,
but did cleave between second and third S phase. This
means that there was an extra round of S phase that
was not followed by cytokinesis. The tetraploidy of these
embryos is ascribed to the extra S phase. In the Opb
eggs, on the other hand, each S phase and cleavage
was observed alternatively, as in normally fertilized
eggs. This maintains the tetraploidy of these types of
eggs.
METAPHASE IS PROLONGED IN SEA URCHIN EGGS
TREATED WITH LOW PH SEA WATER CONTAINING
SODIUM ACETATE.
K. Watanabe, M. S. Hamaguchi, Y. Hamaguchi.
Biol. Lab., Fac. of Sci., Tokyo Inst. of
Tech., Tokyo.
The relationship between mitosis and the
intracellular pH was further investigated
during the first cleavage of sea urchin
eggs. When the eggs of Scaphechinus
mirabilis were treated just before nuclear
envelope breakdown with Ca*°-free sea water
containing 10-20 mM CH3COONa and/or 10-20
mM NH, C1 pH-SW) whose pH was adjusted from
6.2 to 8.0, the intracellular pH was
measured using a fluorescent indicator,
pyranine. The values changed from 7.3
nearly to the pH values of pH-SWs within a
few minutes and became constant. The volume
of the spindle decreased linearly when the
intracellular pH was increased from 6.8 to
8.0. Mitosis progressed normally in the
eggs treated with pH-SWs at pH7.3 or more,
but it didn't progress in those treated
with pH-SWs at pH6.5 or less. When the pH
was lowered by treatment with pH-SWs at
pH6.8 or 7.0, metaphase was prolonged. In
the cases of S. mirabilis and Clypeaster
japonicus this period at pH6.8 was longer
than that at pH7.0. In the case of
Hemicentrotus pulcherrimus, as reported in
MAO, Kyeshs th Wi V7 (1991), the eggs did not
enter anaphase but remained at metaphase
more than 30 min. These results suggest
that the transition from metaphase to
anaphase depends on the intracellular pH.
PSEUDOPODIA FORMING ACTIVITY DURING EARLY
CLEAVAGE STAGES OF SEA URCHIN EMBRYOS.
T.Kominami. Dept. of Biol., Fac. of Sci.,
Ehime Univ. Matsuyama.
After the 6-7th cleavage, blastomeres
of sea urchin embryos become closely
attached one another and change into
columnar configuration. The purpose of
this study is to find out the causal forces
that operate during such a process of
blastulation. The dissociated blastomeres
of mid-to-late stage embryos were found to
form several blebs at the cell surface.
These blebs were retracted in a few
minutes if they failed to attach the the
substratum, and new blebs were formed at
different sites. Blebs which succeeded in
making contact with the substratum became
typical lobo- or filopodia. Detailed
observation revealed that such pseudopodia
forming activity (PFA) appeared after the
7th, 6th and 4th cleavage in macro-, meso-
and micromere derived cells, respectively.
To know the factors which correlate such a
cleavage-dependent phenomenon, size of
blastomeres, nuclei and nucleo-cytoplasmic
volume ratio was measured after each
cleavage. Among these factors, the nucleo-
cytoplasmic ratio was found to closely
correlate the appearance of PFA. The ratio
initially increased logarithmically during
several cleavages, and the ratio was
Maintained at a constant level once the
maximum level had been reached. In any
lineage of blastomeres, the ratio was over
0.1 when PFA appeared.
IMMUNOLOGICAL STUDIES ON EXTRACELLULAR MATRIX
PROTEINS OF SEA URCHINS.
Y.Yokota', V. Matranga*, F. Zito?, M. Cervello*
and E. Nakano”. !Biol. Lab., Aichi Pref. Univ.,
Nagoya. 2Ist. Biol. Sviluppo, Palermo, Italy.
Two extracellular matrix (ECM) proteins of mole-
cular weight 105 kD and 180 kD were isolated from
the sea urchins, Temnopleurus hardwicki, Para-
centrotus lividus and Pseudocentrotus depressus.
A polyclonal antibody against the 105 kD protein
of T. hardwicki was raised and employed in immuno-—
blotting and immunofluorescent microscopy. The 105
kD protein of P. lividus and "echinonectin"” were
cross-reacted with this antibody, whereas the 105
kD protein of P. depressus was cross-reacted weak-
ly. ‘The cross-reactivity of the 105 kD protein
with the antibody seems to have relevance to the
behavior of the protein in the affinity chromato-
graphy on gelatin-Sepharose. Immunof luorescent
microscopy indicated that this antigen is distri-
buted uniformly in the unfertilized eggs but is
localized mainly in the surface area of embryos.
The embryos of T. hardwicki were cultured in the
presence of Fab of the antibody. No significant
effect of Fab on development was observed unti
the gastrula stage but the elongation of skeleton
was inhibited in plutei. These results suggest
that the 105 kD ECM protein is secreted from the
cytoplasm to the outer surface of embryo and plays
some roles in development.
Developmental Biology 1179
PATE OF AN EXTRACELLULER MATRIX COMPONENT
OF FERTILIZED SEA URCHIN (HEMICENTROTUS
PURCHERRIMUS) EGGS
Y.Myotoishil N.Kitai, T.Fukada, S.Ikegami,
K.H.Kato.? IDept. of Applied Biochem.
Hiroshima Wimelavaenr Higashi-hiroshima,
Hiroshima, and 2Bi0l.Lab., Coll. General.
Educ. Nagoya City Univ., Nagoya.
The peptides with the apparent molecular
Masses (Mr) of 99, 52, 49, 39 and 32kDa
are released from the sea urchin blastulae
at hatcing. Components reacting with
antisera raised against the peptide with
the Mr 99 (p99) are concentrated in the
cortical granules of an untertilized egg.
Antigen components translocated into the
fertilization envelope and on the surface
of the plasma membrane. The antigen
components were not removed upon treatment
with Ca?*-,Mg**-free sea water, suggesting
that the components are not hyaline. The
results of the present study demonstrate
the occurrence of a new extracelluler
Matrix component surrounding the outer
surface of the embryo, which is removed at
hatcing.
COLLAGENOUS FIBERS IN THE SEA URCHIN PLUTEI
BLASTOCOEL.
Hee Nakajimal, and Katsuhiko Shimizu! 2.
Dept. of Biol., Keio Univ., Yokohama 223,
2Pusetani Biofouling Project, Yokohama 235.
The existence of collagenous fibers in
the blastocoelic space of the sea urchin,
Hemicentrotus pulcherrimus, Scaphechinus
mirabilis, and Clypeaster japonicus plutei,
was demonstrated by the transmission elec-
tron microscopy. A considerable amount of
fibrils were retained in the blastocoelic
Space of pluteus which were prepared by
quick freeze and freeze substituted tech-
nique. Some of them show cross striation
(60 - 70 nm periodicity) and had a width of
20 nm. They were morphologically resemble
to the type I collagen. Fibroblastic
mesenchymal cell in the blastocoel of
pluteus arm spun out fibers from the cyto-
plasmic pit.
Two types of collagen disrupt agents,
B-aminopropionitrile and aa'-dipyridyl,
dose dependently inhibited further exten-
sion of the arms of C. japoinus prism and
pluteus larvae. Collagen fibers in the
blastocoelic space of pluteus may contrib-
ute to the morphogenesis of pluteus.
It might be still remain to be further
verified whether the blastocoelic meshwork
of rapidly frozen and freeze substitution
of the Srtongylocentrotus purpuratus blas-
tula reported by Cherr et al.(1992) were
due to ice crystals with the surrounding
eutectic phase of blastocoelic substances.
ULTRASTRUCTURE OF THE AXIAL ROD COMPOSED
OF A BUNDLE OF ACTIN FILAMENTS IN ABALONE
SPERM REVEALED BY QUICK FREEZE TECHNIQUE.
Yoo sieOiyiai mance Nee leers akan, By OM | IhENS 5
Wayo Women's Univ., Chiba.
eee
An axial rod in abalone (Haliotis
discus) sperm is a motile structure com-
posed of a bundle of actin filaments. The
axial rod elongates anteriorly to form an
acrosomal process during the acrosome
reaction. The ultrastructure of the axial
rod was examined using quick freeze tech-
nique. Thin sections of quick freeze and
freeze-substituted sperm revealed that the
actin filaments within the axial rod are
hexagonally packed in a paracrystalline
array almost through its entire length
With an average center-to-center spacing
of 12nm. Periodic transverse bands were
also observed across the actin filament
bundle. Quick-freeze deep-etch analysis
provided the three-dimensional view of the
axial rod. Actin filaments exhibiting 5.5-
6nm spaced striations were observed to run
in parallel with each other. Cross-
bridging materials were displayed between
adjacent filaments. These results suggest
that the actin filaments in the axial rod
are probably held together by regular
cross-bridges to form paracrystalline ar-
ray, and also cross-linked by 3-4nm fila-
ments to the lateral membrane which
closely surrounds the anterior half of the
actin filament bundle.
ACROSOME REACTION AND SPERM PENETRATION IN THE
BIVALVE. LATERNULA LIMICOLA.
K. Hosokawa and Y.D.Noda“. Biol. Lab., Tokyo
Dent. Coll., Chiba. ?Biol. Inst., Fac. Sci., Ehime
Univ., Matsuyama.
Acrosome of the mature sperm of lLaternula
limicola are presented at the posterior end of the
mitochondria in the mid-piece. To ascertain the
role of the posterior acrosome, the process of
acrosome reaction and sperm penetration were
observed by an electron microscopy.
The first step of the acrosome reaction is a
pit formation at the posterior tip of the acrosome
vesicle. The second step takes place at the ab-
dominal part of the acrosoinmal vesicle, namely, the
sperm plasm membrane and the outer membrane of the
vesicle burst open at the abdominal part and fuse
with one another. In the third step, membrane re-
gression occurs immediately follow in the membrane
fusion. As a result, the interior membrane of the
vesicle replaces the exterior membrane of the sub-
acrosomal region and obtains the capacity to fuse
with the oolema. In the fourth step, the replaced
membrane of the subacrosomal region makes contact
with the tip of one of the microvilli remaining in
continuity with the oolema and rapidly fuses with
microvilli membrene. A fertilization cone is
formed at the point of the membran contact.
Finally, sperm penetration begins from the
posterior ventral region of the sperm. The sperm
organelles incorporated into the fertilization
cone preceed the microfilament, follow by five
mitochondria and the sperm nucleus is the last to
the enter the ooplasm.
This form of sperm penetration is a new mode of
fertilization.
1180 Developmental Biology
MORPHOLOGICAL CHANGES OF HAMSTER ISOLATED
SPERMATOGENIC CELLS AFTER ELECTROFUSION
WITH HOMOLOGOUS OVA.
N. Usuil, a. Ogura? and R. Yanagimachi3.
lpept. of Anat., Teikyo Univ. Sch. of Med.,
2Dept. of Vet. Sci., Natl. Inst. of Health,
Tokyo and 3Dept. of Anat. & Reprod. Biol.,
Univ. of Hawaii Sch. of Med., Hawaii.
When an electrofusion pulse is applied,
hamster mature ova fuse with isolated round
spermatids and most of them develop to the
2-cell stage (Ogura et al, '93, Zygotes, in
press). To elucidate the effect of ooplasm
on the nuclei of spermatogenic cells (SCs),
ova fused with isolated SCs were examined
by means of thin sectioning.
At 3-5 h after fusion, the SC nuclei re-
mained almost unchanged in their shapes and
sizes; the acrosomal vesicle was found at-
tached to the spermatid nucleus. Detailed
observations, however, revealed structural
modifications in the spherical nuclei of
early spermatids (the Golgi and cap phases)
as well as spermatocytes. As female pro-
nucleus developed, these nuclei gained the
characteristics of the pronucleus: (1) prom-
inent spherical nucleoli, (2) the so-called
blebbing of the nuclear envelopes and (3)
annulate lamellae in their vicinity.
Perhaps, MPF did not affect the SCnuclei
because it had disappeared before intermin-
gling of the SC cytoplasm with the ooplasm,
whereas cytoplasmic factors responsible for
pronuclear development remained and induced
modifications in the spherical SC nuclei
comprising dispersed chromatin.
BINDING OF BUFO JAPONICUS SPERM TO THE
VITELLINE COAT AND ITS RELATION TO
FERTILIZATION.
S. Omata and Ch. Katagiri, Zoological Institute, Faculty
of Science, Hokkaido University, Sapporo
Dejellied uterine eggs (UE) of Bufo japonicus are
not fertilized in 1/20 De Boer's solution (1/20DB), but are
fertilized in the reconstituted salt solution (RSS) which
mimics the ionic conditions in jelly envelopes.
Concomitant with this difference in fertilizability, the
Vitelline coat (VC) of the eggs inseminated in RSS had
more than 20 times as many sperm bound to those in
1/20DB. Determination of the number of sperm bound to
a unit area (0.2mm*) VC indicated that the sperm
binding to UEVC (a) is dependent on the concentrations
of both Ca** and Mg** in RSS, (b) is lost after activation
of eggs in 1/20DB but not in DB, and (c) is competitively
inhibited by the VC materials solubilized by SDS. The
rate of binding was extremely low to the VC of coelomic
eggs (CEVC), but increased to a level equivalent to that
of UEVC after the treatment of coelomic eags with the
extract of pars recta (PR), the upper 1/20 portion of
oviduct, or with trypsin. This effect of PR was ascribable
to the trypsin-like enzyme which caused a limited
proteolysis of CEVC. Observations on transmission
electron microscopy revealed that the sperm bound to
VC were not acrosome-reacted. These results suggest
that the proteolysis by oviducal PR protease enables VC
bound to fertilizing sperm before the occurrence of
acrosome reaction.
SCANNING ELECTRON MICROSCOPIC STUDIES ON
THE ACROSOME REACTION INDUCED BY JELLY
LAYERS AND JELLY EXTRACTS IN THE NEWT.
K. Onitake, I. Matsumoto and N. Nishiyama.
Dept. of Biol., Fac. of Sci., Yamagata
University, Yamagata.
When the oviducal eggs were inseminated,
all sperm remaining in the capsular cham-
ber caused the acrosome reaction (AR) and
the acrosomal caps disappeared following
exposure of their perforatoria. Further-
more, sperm incubated with jelly extracts
(JE) caused acrosome reaction in very high
rate. In the present study, we examined a
process of acrosome reaction of the newt,
Cynops pyrrhogaster, by JE in comparison
with sperm passing through jelly layers
by SEM. When spermatozoa were incubated
with JE, acrosome reaction was caused
within 60 sec. Process of acrosome re-
action was as follows: (1) the disruption
of acrosomal cap began within 30 sec, and
(2) disrupted acrosomal cap was stripped
and the perforatorium was exposed within
60 sec. On the other hand, the disruption
of acrosomal caps of sperm passing through
the jelly laysers occured within five min-
utes. Some of sperm observed in Jl to J4
caused acrosomal reaction completely,and
the other was on the way of AR. Sperm just
after passed through Jl(inner most) showed
complete AR or incomplete disruption of
acrosomal cap. These results may suggest
important role of jelly on the fertiliz-—
ation and gentle polyspermy block in the
newt which is physiological polyspermy.
ON THE ROLE OF ACROSOME REACTION INDUCING
FACTORS FROM EGG JELLY IN THE NEWT
FERTILIZATION
S. Nishida and K. Onitake. Dept. of Biol.,
Fac. of Souls Yamagata University,
Yamagata.
We have reported that the extracts from
the newt, Cynops byrrhogaster, jelly
layers(jelly extracts:JE) had the acrosome
reaction(AR) inducing factors, and they
were synthesized at the specific region of
the oviduct in the HCG-injected ovulating
female. In the present studies, we
investigated the role... jo& AR-inducing
factors on the fertilization using the
antibodies against JE. When JE were
incubated with anti-JE antibodied or
papain digested univalent antibodies, AR
inducing activities were neutralized. AR
could not be induced completely by JE
incubated with univalent antibodies.
Moreover, the fertilization ratio of
uterine jelly eggs was reduced strongly
when they were treated with univalent
antibodies and inseminated artificially.
In addition, the oviducal tissues obtained
from ovariectomized newt for 6 months were
not stained with anti-JE antibodies by the
indirect immunofluorescent histochemistry.
These results suggests that AR inducing
factors in the jelly layers were closely
related with fertilization in vivo, and
the synthesis of them was controlled by
the hormone produced in the ovary.
Developmental Biology 1181
POLYSPERMY BLOCK IN THE LAMPREY, LAMPETRA
JAPONICA EGGS
W. Kobayashi’, Y. Baba?, and T. Shimozawa?
Zoological Institute, Faculty of Science, Hokkaido
University, Sapporo.
21 ab. of Neuro—Cybernetics , Res. Inst. for
Electronic Science, Hokkaido University, Sapporo.
Sperm entry in the lamprey eggs is restricted toa
region near the animal pole (sperm entry site, SES).
Upon insemination of the eggs, the membrane potential
rapidly sifted from -12 mV to +36 mV (fertilization
potential). Sperm were unable to fuse with the eggs
voltage clamped at more than +10 mV, indicating that
the fertilization potential participates in the fast block
against polyspermy. Duration of the effective fast block
was about 160 seconds. Previous studies showed that
sperm-—egg fusion has been established at the tip of the
acrosomal filament of sperm although its nucleus
remains outside the egg envelope, and that sperm-egg
fusion is prerequisite to the passage of sperm through
the envelope. In this study, transmission electron
microscopic observations revealed that the acrosomal
filaments were unable to touch the ooplasmic surface
after separation of the egg envelope from the ooplasmic
surface. This fact indicates that formation of the
perivitelline space at the SES is responsible for a
permanent block against polyspermy. Establishment of
the permanent block was about 160 seconds after
initiation of the fertilization potential. These results
suggest that the permanent block is not quite fast
enough to replace the electrical block against
polyspermy, and do not rule out the occurrence of
another intermediate block.
ACTIVATION OF STARFISH EGGS BY CAFFEINE: Ca2*-
RELEASE, CORTICAL GRANULE EXOCYTOSIS AND
OXYGEN CONSUMPTION.
S. Nemoto!, K. Kato*, A. Hino, T. Mohri, Y. Hamaguchi4
and H. Nakagima>. !Tateyama Marine Lab.,Ochanomizu
Univ., *Nagoya City Univ., 7Kanagawa Univ., “Tokyo Univ.
of Technol. and >Aichi Univ. of Edu., Japan.
As we previously reported, caffeine activates both immature
(GV stage) and maturing (after GVBD) oocytes. In the present
study, we determined Ca2* release on caffeine activation of both
immature and maturing oocytes of Asterina pecunifera using
Fura-2 in relation to the formation of the fertilization envelop
(FE), cortical granule (CG) exocytosis and consumption of
oxygen. There was no difference between maturing and
immature oocytes in caffeine concentrations required for
inducing Ca2* release comparable to that in normal fertilization
(10 mM or higher). Immature oocytes, however, formed only a
partial FE, in contrast with maturing oocytes that did fully
elevated the FE and a significant number of CGs were observed
to remain unbroken in immature oocytes. A transient increase in
Oxyge consumption (burst) was small in immature oocytes as
compared with that in maturing oocytes. These differences
between both oocytes were the same in A 23187 activation. The
present results suggest that contact of CGs with egg cell
membrane is established by the time of GVBD after the
Teinitiation of meiosis and that Ca**-release Ca2+ machinery
may participate in Ca** increase on fertilization.
EFFECTS OF H-7 AND W-7 ON CHANGES IN THE
RATE OF OXYGEN CONSUMPTION AND THE CONTENT
OF ARGININE PHOSPHATE FOLLOWING TREATMENTS
WITH ACTIVATING REAGENTS IN SEA URCHIN
EGGS
M.K. Kojima, N.Iishizawa, H.Hirai and
S. Nakamura. Dept. of Biol., Fac. of Sci.,
Toyama Univ., Toyama
We reported that various kinds of activat-
ing reagents, such as A23187, procaine,
ammonia and TPA, can induce both an en-
hancement of oxygen consumption and an in-
crease of the content of arginine phosphate
(ArP) in unfertilized sea urchin eggs. And
we suggested a possibility that metabolic
stimulation by these reagents have a close
relation with a release of cytoplasmic Ca?*
and a rise of cytoplasmic pH. Therefore,
in the present study, it was determined
whether W-7, a calmodulin inhibitor, and
H-7, an inhibitor of protein kinase C, can
suppress such a rise of 0, consumption or
of the ArP content following treatments
with above-mentioned activating reagents.
Thus, it was revealed that an enhancement
of O21 consumption is cancelled only by com-
bining treatments of TPA with H-7, and by
treatments combined A23187 with W-7, but
not by combination of H-7 or W-7 and pro-
Caine or ammonia. On the other hand, H-7
has no effect on increased ArP content
after treatments with the above-mentioned
four activating reagents. Similar results
are obtained by treatments combining W-7
with those activating reagents.
POLARITY OF UNEQUAL CELL DIVISION IN
GRASSHOPPER NEUROBLASTS.
K. Kawamura. Lab. of Biol., Rakuno Gakuen
Uviv., Ebetsu, Hokkaido. ¢
Grasshopper neuroblasts repeat unequal
cytokinesis along the dorso-ventral axis of
the embryo. They produce a small ganglion
cell to the dorsal side. Each neuroblast
is accompanied with one or more cap cells
on the ventral side surface and a column of
ganglion cells extending to the dorsal
side. When the spindle body at metaphase
was rotated approximately 90° by a
microneedle, the rotated spindle returned
to the original direction by autonomous
turning. Therefore, the polarity of
neuroblasts have been already determined by
metaphase. In the present study, various
microdissections such as the spindle
rotation, the spindle shifting, the bending
of half spindle, the destruction of
surrounding cap cells or ganglion cells,
were performed in dividing neuroblasts.
The subsequent cell divisions in these
microdissected neuroblasts were traced in
order to analyze the polarity of unequal
cell division. The results obtained here
suggests that the preceding division axes
participate in determination of the
division axis in subsequent division. The
cortical differentiation concerning the
polarity seems to be commenced at late
anaphase of the preceding division, and to
be established only in the condition that
cytokinesis is completed.
1182 Developmental Biology
BEHAVIOR OF CENTRIOLE PAIRS IN YOUNG
NEUROBLASTS OF CHORTOPHAGA.
N. Yamashiki. Biology, Rakuno Gakuen Univ.,
Ebetsu, Hokkaido.
Neuroblasts in the grasshopper embryos
repeat unequal cell division that produces
a small daughter cell (a ganglion cell
(GC)) and a large daughter neuroblast (NB).
In the previous electron microscopic
studies, centriole behavior was observed by
uSing 16-17 day old embryos in which
mitotic activity of NBs is quite high. The
centriole pair in the centrosome were close
together at both spindle poles by
metaphase. In late anaphase when the
spindle took an eccentric position close to
GC-side, the centriole pairs at the two
spindle poles behaved differently. The
pair at NB-side pole were separated, while
the pair at GC-side pole remained close
together. In this study, centriole
behavior was observed in young NBs from 8-
10 day old embryos. The young NBs have
experienced one or two mitosis after their
banotchis In metaphase, the both centriole
pairs were close together, as observed in
16-17 day old embryos. However, centriole
behavior in late anaphase was different in
the young NBs. The centriole pairs at the
both spindle poles were slightly separated,
and no differential behavior was detected.
The differential behavior of centriole
Pairs between the two spindle poles seems
to be acquired with ageing of the NBs.
FLUORESCENE MICROSCOPIC OBSERVATIONS OF
CONE MOSAIC OF THE RETINA OF MEDAKA.
Y.Nishiwakit, T. Oishi, s.Horiuchi2,
T.Morita and F.Tokunaga”. IDept. of
Biol., ac. of Sci., Nara Women's Univ.,
Nara, ME Ne, CRE iil, (@WwlS oir (en.
JaolbKeE , eyeVol “dKeyoe > ee Uet@l,, jee Cr Sel. -
Osaka Univ., Toyonaka.
The distribution of cones in the retina
of teleost has been observed as two
organizational patterns, a square mosaic
and a row pattern. In the medaka, three
kinds of cones form a square mosaic
pattern which consists of two rows, rows
of zig-zagging double cones and rows of
alternating short and long single cones.
In the section of the retina of adult
medaka, some kinds of cells could be
detected by its autofluorescence without
staining, using a fluorescence micro-
scope, equipped with blue excitation
filter. We discriminated each type of
cones by their morphological differences.
While this mosaic pattern was hardly
observed in the pigment layer with
conventional light microscope, these cones
were clearly identified by their emission
of autofluorescence against the pigment
layer as a background. The same cone
mosaic pattern could be detected in
growing retinae after hatching.
This autofluorescence method is useful
to analyze the pattern of cone mosaic in
the retina of the medaka.
FORMATION AND DISTRIBUTION OF VISUAL
CELLS IN CHICKEN RETINA
S. Horiuchi’, H. Kobayashi'?, F. Tokunaga?, Y. Fukada‘,
Y. Shichida*, T. Yoshizawa’, T. Morita! and Y. Koshida!.
1Dept. of Biol., Coll. of Gen. Educ., Osaka Univ., Toyo-
naka, *Dept. of Biol., Fac. of Sci., Osaka Univ., Toyonaka,
‘Dept. of Biophys., Fac. of Sci., Kyoto Univ., Kyoto, *Dept.
of Appl. Phys. and Chem., Univ. Electro-Communications,
Tokyo. Present adress: Dept. of Biophys. Engineer., Fac.
of Engineer. Sci., Osaka Univ., Toyonaka.
To investigate the histogenesis of chicken retina, espe-
cially the formation of the distribution pattern of rod and
cone cells, we applied monoclonal antibodies (mAb) raised
against cattle rhodopsin (Tokunaga et al., Zool. Sci. 6:167-
171, 1989) and raised against chicken iodopsin and R-photop-
sin (Shichida et al, Exp. Eye Res., 48:281-293, 1989) for im-
munohistochemical studies to the developing retinae of chick
embryos. Using FITC indirect immunofluorescence method,
FITC positive visual cells could be detected at first in 16-day
embryo by anti-cone visual pigment mAb and 3 days later
rod cells could be detected by anti-cattle rhodopsin mAb.
No particular regularity of distribution pattern of rod and
cone cells was recognized in developing chick embryo retinae.
These mAbs are useful marker to examine which of the visual
cells differentiate formerly in the juvenile retinae.
MONOCLONAL ANTIBODIES SPECIFIC FOR PHOTO-
RECEPTOR AND BIPOLAR CELLS IN THE NEWT
RETINA.
F.Maruo, Y.Sakaki, Y.Kaneko and T.Saito.
Inst. Biol. Sci.«,. Univ” Of eetsuncmbay
Tsukuba.
Regeneration of the adult newt retina follow-
ing its destruction provides us with a useful
system for understanding the genesis of neural
circuitry in the vertebrate central nervous
system. In this study, we prepared monoclonal
antibodies (MAbs) against neural retinas of
adult newts, Cynops pyrrhogaster, to obtain
markers of cell types for analysing retinal
development and regeneration. MAbs were
screened with immunofluorescence staining
using frozen sections of retina. RB-1 MAb
stained a subtype of bipolar cells and the
cone-type photoreceptor cells. During normal
development, RB-1 MAb first reacted with
photoreceptors in the retina before segregation
into distinct synaptic layers at stage 35, and
then reacted with both photoreceptors and a
bipolar cell subtypes in the retina with
segregation into the synaptic layers at later
stages. RB-1 MAb did not stain other type of
cells during retinal development. RB-2 MAb
stained both cone and rod photoreceptor cells
and a subtype of bipolar cells. RB-2 MAb bound
to a single band of 26 kDa in the immunoblot
analysis. This band was also reacted with
antiserum against recoverin, which is a Ca*’-
binding protein found in photoreceptors of
bovine retina. RB-1 and RB-2 MAbs could be
used as differentiation markers for specific
retinal cells.
Developmental Biology 1183
EFFECTS OF SAPOSINS ON THE DEVELOPMENT OF
ORGANOTYPIC CULTURE OF NEWBORN MOUSE
CEREBELLUM.
D.Satomil, Y.Kishimoto2. 1Dept. of Biol.,
Coll. of Arts and Sci., Univ. of Tokyo,
Tokyo, 2Ctr for Molec. Genet., Univ. of
Calif., San Diego, La Jolla, USA.
Saposins(A,B,C and D),sphingolipid act-
ivator proteins,are all small heat-stable
glycoproteins derived from a common precu-
sor, prosaposin. Recently, it was found
that saposins bind strongly gangliosides
and are present at high concentrations in
mervous tissues. To investigate the roles
of saposins on neuronal development, we
used cerebellar culture system. Saposin A
or B was added to feeding medium from 2
days in vitro(2 DIV). In saposin A treated
explants, ependymal formation was observed
at 8 DIV. Myelination did not occur during
incubation. A number of large flat cells
with few long processes were present char-
acteristically in the circumference of 15
and 22 DIV explants. The levels of 2',3'-
eyclic nucleotide 3'-phosphodiesterase
(CNP) and glutamate decarboxylase(GAD)
were low during incubation. In saposin B
treated explants, morphologically ependy-—
mal formation and myelin formation were
observed. The activities of CNP and GAD
increased nearly same manner as those of
control.These observations indicate that
biological function of saposin A and B is
Significantly different despite their sim-
ilarity in structure.
ANALYSIS OF CYTOSKELETON OF GnRH NEURONS
MIGRATING ALONG OLfACTORY NERVE IN CHICK
EMBRYO.
M.Miyakawa, K.Wakabayashi_ and Y.Arai.
Dept; Anat - Juntendo Univ.Sch.Med., Tokyo
and ~Inst.Endocrinol.Gunma Univ.,Maebashi.
In recent years a considerable body
of evidence has been accumulated suggest-
ing the migration of GnRH neurons from the
olfactory placode to the brain along the
olfactory nerve in chick embryos. To know
the possible mechanism of neuronal migra-
tion, cytoskeletal elements of these neu-
ronal substrates were studied. The locali-
zation of actin filaments was observed by
rhodamine-labelled phalloidin. Monoclonal
antibodies to GnRH and d&-tubulin were
used to detect the presence of GnRH and
microtubules, respectively by immunohisto-
chemical methods. By double staining of
GnRH and 4-tubulin, GnRH-immunoreactive
(IR) neurons were observed to be closely
associated with the olfactory nerve axons
from the olfactory epithelium to the
brain. Double staining of actin and GnRH
or tubulin revealed that the nerve associ-
ated with GnRH-IR neurons showed plenty of
phalloidin-labelled actin compareing to
the other nerve bundles. The coexistance
of GnRH-IR cells and abundant actin fila-
ments was marked at the sites of the emer-
gence from olfactory epithelium and the
entrance into the brain. The actin-rich
processes of GnRH-IR neurons may play an
important role in the neuronal migration
scaffolded by the olfactory nerve.
EXPRESSION OF SC2 ANTIGEN ON FASCICULATING
PRIMARY AFFERENT FIBERS IN THE DORSAL
FUNICULUS OF CHICK EMBRYO SPINAL CORD.
T.Shiga’. Te Shire "and Mi, Tamale.
"Dept of Anat., Yamagata Univ., Yamagata
and Dept. of Neurosci. & Immunol.,
Kumamoto univ., Kumamoto.
We have been investigating the
mechanisms of pathway formation of spinal
primary afferent axons (PAAs) in chick
embryos. In the present study, we
examined the expression of SC2 antigen
(specific cell surface molecule for dorsal
root ganglion (DRG) cells and PAAs, Tanaka
and Obata ‘84), using immunocytochemi cal
method. Surfaces of DRG cells and PAAs
became SC2 positive on embryonic day 3
(stage 19-20 of Hamburger and Hamilton,
monly) when the earliest growing PAAs
entered the spinal cord from DRG.
Thereafter PAAs ran rostrocaudally in the
dorsal funiculus in a fasciculated manner
These PAAs expressed strongly SC2. On
day 6 when the first PAAs entered the
dorsal horn through the dorsal funiculus
without fasciculation, PAAs in the dorsal
funiculus and dorsal horn became weakly
SC2 positive or negative, respectively.
In contrast, PAAs expressed neuron-glia
cel | adhesion moleculue (Ng-CAM),
irrespective of the state of their
fasciculation. Therefore, these results
suggest that SC2 may be involved in the
fasciculation of PAAs.
NEURITIC PROCESS OUTGROWTH FROM CALCITONIN-
PRODUCING CELLS IN VITRO.
I.Nishiyama!, T.Seki?, T.Oota3, M.Ohta* and M.Ogiso‘.
Dept. of Pharmacol., Teikyo Univ. Sch. of Med.,
2Dept. of Anat., Juntendo Univ. Sch. of Med., 2Iso-
tope Cent., Tokyo Univ. of Agri., and *Dept. of
Physiol., Toho Univ. Sch. of Med., Tokyo.
A calcitonin-producing cell (C-cell) is one of the
endocrine derivatives of the neural crest, and it
shares several properties with the neuron. The
phenotypic plasticity of the C-cells was examined in
culture systems. The ultimobranchial bodies, the
embryonic organs composed mainly of C-cells, were
isolated from 16-day rat fetuses and cultured in
serum-supplemented conventional medium. At Day 4
in vitro, the cells were subjected to double immuno-
staining using an anti-calcitonin antiserum and a
monoclonal antibody (MAb) 12E3 which reacts specif-
ically with polysialic acid portion of embryonic
neural cell adhesion molecule. Most of the C-cells
were polygonal or ovoid in shape, and few of them
were positive to MAb 12E3. When a protein kinase
inhibitor H-7 (30 to 70 M) was added to the cul-
ture medium at Day 3 in vitro, many C-cells were
found to extrude long neuritic processes and to
exhibit intense immunoreactivity to MAb 12E3 within
24 h. The longest process reached 300 pm in
length. Some processes had growth cones at their
terminals. The effect of H-7 was apparently antago-
nized by 40 nM okadaic acid, a potent inhibitor of
protein phosphatases. Practically the same results
described above were observed in rMTC 6-23 cells,
a C-cell line derived from rat medullary thyroid
carcinoma. These results suggest the involvement
of some protein kinase(s) in morphoregulatory sys-
tem in the C-cells.
1184 Developmental
SNAIL-LIKE MOVEMENT OF THE CHICK PRE-
STREAK STAGE EPIBLASTIC CELLS IN VITRO
Ryuji Toyoizumi and Shigeo Takeuchi. Dept. of
Biol. Sci., Fac. of Sci., Kanagawa Univ.. Hiratsuka.
To understand the mechanism how the epiblastic
cells (EC) of amniotes move to form the primitive
streak, we dissected the cells from chick pre-streak
stage embryo, dispersed into single cells, cultured
with our conditioned medium, and observed their
behavior in vitro .
On the substratum coated with laminin (LN) gel,
EC attached in a spherical form, and protruded one
or a few very long stick-like lobopodia extending up
to 20 um, whose figures looked like snails. But they
hardly locomote on it. After 24 hr in culture. each
cell dissolved the LN gel beneath it to make a hole
and settled in it. On the substratum lined in parallel
with fibrous LN gel, EC extended the long lobopodia
along the lines. But, also in this case, they hardly
locomoted. Single epiblastic cells poorly attached to
the substratum coated with fibronectin (FN).
However, on the substratum lined in parallel with LN
gel supplemented with a small amount of FN, they
protruded the long lobopodia along the lines from
their spherical cell bodies, and locomoted
unidirectionally along it.
Substratum containing both LN and FN, somehow
align in the blastoderm might support the migration
of EC to form the pnmitve streak in situ.
HOW DOES REPLICATION TIMING OF
SPECIFIC DNA ESTABLISH IN
DEVELOPMENT. - A STUDY OF IMAGE
ANALYSIS.
S. Tanaka, T.Tsuji, Y. Kawasaki and T. Saitoh
Mitsubishi Kasei Institute of Life Sciences. 11
Minamiooya, Machida-shi, Tokyo.
Replication timing of genes and
subchromosomal segments in differentiated cells
is fixed throughout successive cell cycles and
closely related with their states of activities.
Although replication timing of respective
replication domains (replicons) is presumed to
be established during embryonic development,
thorough studies are lacking and _ its
developmental significance is uncertain.
Present study is aimed to exploit a method
of image analysis for measurements of replication
timing of specific DNA of avian scale dermal cells
during their initial morphogenesis. Experimental
conditions and relevant methods were examined
in various points such as follows: doses and time
intervals of double labeling of DNA with
bromodeoxyuridine (BrdU) and iododeoxyuridine
(IdU) during S phase in jn vivo system,
immunocytochemical detection of BrdU and IdU,
multiparametric microfluorometry of DNA content,
amounts of BrdU and IdU of individual cells, image
capturing of double stained replicons by confocal
laser scanning microscope and image processing
for identification and characterization of replicons
classified by kinds of combinations of the labeling.
Biology
ROLES OF HEMOPOIETIC CELLS IN LYMPHOID
FOLLICLE FORMATION IN THE BURSA OF FABRI-
CIUS. Wer SiloaiGyyprirent Dept. Biol., Fac.
Sci., Shizuoka Univ., Shizuoka.
Lymphoid follicles develop from the
epithelial part which hemopoietic cells
invade in the bursa of Fabricius of the
avian embryo. The present study was under-
taken to reveal what roles hemopoietic
cells play in the lymphoid follicle forma-
tion of the bursa.
Bursas of 6-day quail and 7-day chick
embryos which were not colonized by hemo-
poietic cells were transplanted onto the
chorioallantoic membrane (CAM) of chick or
quail embryos. The chick bursas were also
co-transplanted with the axial mesenchyme
containing hemopoietic cells onto the CAM
of quail embryos. The transplants was
examined histologically.
Quail bursas developed abundant lymphoid
follicles containing host hemopoietic cells
when transplanted on the CAM of either
chick or quail embryos. In contrast, chick
bursas hardly developed lymphoid follicles
when transplanted on the CAM of quail
embryos. Co-tranplantation of chick bursas
with the axial mesenchyme produced lymphoid
follecles in the transplants. These results
suggest that chick hemopoietic cells are
necessary for chick bursal follicle forma-
tion. Invasion of hemopoietic cells in the
bursal epithelium may be indispensable for
the lymphoid follicle formation in addition
to the bursal epithelium and mesenchyme.
AN ELECTRON-MICROSCOPIC STUDY ON THE EARLY
DEVELOPMENT OF THE FRESHWATER PLANARIAN
BDELLOCEPHALA BRUNNEA, WITH SPECIAL
REFERENCE TO THE FORMATIONS OF GROUP AND
SYMCYTIUM OF YOLK CELLS.
W. Teshirogi, H. fujino, S. Sugimori and
S. Ishida. Dept. of Biol., Fac. of Sci.,
Hirosaki Univ., Hirosaki.
The egg of freshwater planarians is a
composite egg, composed of several egg
cells and numerous yolk celis. The yolk
cells around the egg cell surrounded the
egg cell radiately, shortly after
egg-laying. These yolk cells then formed
the syncytium. Seilern-Aspang (1958) and
Koscielski (1966) have assumed that the
grouping substance released from the egg
cell ' at 1-cell stage, and syncytial
substance released from blastomeres. This
time, we have examined these in embryo at
1-to 32-cell stage by electron microscopy.
The cytoplasm of egg cell is filled with
ER of vacuole type and free ribosome, and
the ER vacuole are released outside of the
egg. This is regarded to be closely
related with grouping substance. After
that, not only ER vacuoles but also plenty
of vacuoles originated from mitochondria
are released into the syncytium. We
believe that these vacuoles are related
with syncytial substance. The activities
of these releases are high at 4-to 8-cell
stages and it falls remarkably at the 16-
ceil stage. The lysosomes and
autophagosomes that appear in the yolk
cells probably bring about autodigestion
of the cytoplasm.
Developmental Biology 1185
CHANGES OF CELL DISTRIBUTION AND STRUCTURE
IN DIFFERENT BLASTEMAL REGIONS OF THE
PLANARIANS.
I.Hori. Dept.of Biol.,Kanazawa Med.Univ.,
Uchinada-machi, Ishikawa.
It is a well-established fact that a head
regenerates at different rate from various
levels along the longitudinal axis of the
planarian body. This study was designed to
examine this phenomenon cytologically. Two
species, Dugesia and Phagocata, were used.
The animals were transected respectively
at four levels; just behind eyes(region A),
pre-pharyngeal(B), post-pharyngeal(C) and
tail(D) regions. The 3rd and 5th-day
blastemata were obtained from each piece.
In region A, cell density in the blastema
decreased significantly between 3rd and
5th day while it did not change in regions
C and D. This was in accordance with the
differentiation rate of regenerative cells.
One of the morphological features of well-
differentiated cells was numerous elongated
cell processes with microfilaments. When
cytochalasin B was administered in the late
phase of regeneration, these processes were
inhibited indicating that a scaffold of
differentiating cells within the blastema
is produced by such processes which contain
microfilaments. Staining with ruthenium red
revealed a large quantity of the reticular
filaments surrounding these processes. In
regions, C and D, the less differentiated
cells occupied the blastema but they had
few prominent cell processes. The extra-
cellular matrix was organized poorly.
THE OBSERVATION OF GASTRODERM-FORMATION BY
IMMUNO ELECTRON MICROSCOPY IN THE
FRESHWATER PLANARIAN PHAGOCATA VIVIDA-
T. Kuwahara, S. Ishida and W. Teshirogi.
Dept. of Biol., Fac. of Sci., Hirosaki
Univ., Hirosaki.
Monoclonal antibody (mAb) 6@12B-D
reacted specifically to gastrodermal
phagocytic cells in Phagocata vivida. We
reported with light microscope that this
antibody have reacted to differentiating
cells associated for the repair of
gastroderm (1991). This time, we attempted
to detect the specific antigen of mAb
§12B-D by immuno electron microscopy using
ABC and PAG methods in phagocytic cells.
And we also observed undifferentiated
cells (neoblasts) and differentiating
cells into gastrodermal cells.
The specific binds of colloidal gold
were recognized lipid droplets in
phagocytic cells. In differentiating
cells, the specific binds were also
recognized the newly-formed lipid
droplets.
PHARYNX-FORMATION IN REGENERATION OF THE
FRESHWATER PLANARIAN PHAGOCATA VIVIDA.
K. Onodera, S. Kobayashi, S. Ishida and W.
Teshirogi.
Dept. of Biol., Fac. of Sci., Hirosaki
Univ., Hirosaki.
Some histilogical investigations have
revealed that regenerated pharynx
including its inner epithelium are derived
from gastrodermal cells (Teshirogi (96)Asai
1991 and others),
This time, pharynx-formation was
observed using monoclonal antibody (mAb)
612B-D which react specifically to
gastrodermal phagocytic cells of Phagocata
vivida,immuno histochemically. The antigen
of mAb was recognized in the pharyngeal
inner epithelium of the basal region of
differentiating pharynx.
As a conclusion, it was suggested that
the new pharyngeal inner epithelial cells
originate from old gastrodermal cells.
We have also obtained a mAb which
recognize the materials existing
abundantly in the pharynx.
PRESENCE OF RHODOPSIN-LIKE PROTEINS IN
THE PLANARIAN (Dugesia japonica
Japonica).
Ss Nakamura! , T. Shinozawa“, S. Ishida!
1:Dept. of Biol., Fac. of Sci., Hirosaki
Univ., Hirosaki. 2:Dept. of Biol. and
Chem. Eng., Fac. of Eng., Gunma Univ.,
Kiryu.
Planarian has two sensory organs. One
is visual system which consisted of
pigment-cup ocells and the other is
auricles as tactile system and chemore-
ceptor.
In Western blot analysis of the
planarian head, anti-frog-rhodopsin
rabbit IgG recognized the presence of
rhodopsin-like proteins with apparent
molucular weight of 62,000 and
65,000(Fujita et al. 1991).
In this report, we examined the
localization of rhodopsin-like proteins
by fluorescent antibody technique and
Protein-A gold method. Rhodopsin-like
proteins were locarized in the membrane
of microvilli of the eyes. We considered
that the auricles were associated with
not phototaxis but a different role to
the light stimulation.
1186 Developmental Biology
INTERACTION BETWEEN EYES AND BRAIN OF
PLANARIAN (Dugesia Japonica), ANALYZED BY
THE ANTI-VISUAL CELL SPECIFIC ANTIBODY.
--FORMATION OF SUPPLEMENTARY EYES AND THEIR
PROJECTION ONTO VISUAL CENTERS.
F.Sakai,K.Agata,K.Watanabe, Lab.of Regen-
eration Biology, Fac.of Sci., Himeji Inst.
of Technology. Ako, Hyogo, 678-12 Japan.
Supplementary eyes of planarian occur
spontaneously and experimentally. Their
visual system were examined to analyze
mechanism of eye formation and interrela-
tion between eyes and brain. A monoclonal
antibody was used to visualize a whole
image of planarian visual cells by fluores-
cent antibody technique. With a binocular,
supplementary eyes were classified into two
types: separate eyes and partially joined
eyes(two pigment eye cups with a single
mass of visual cells). However, microscopic
observation showed similarity between two
types. Optic nerves of supplementary and
original eyes run along separate pathways
towards brain. Projection from the fore
eye was always located more medially than
that of the hind eye. Two optic nerves (on
the one side) shared same projection cen-
ters in some cases, and did not in others.
Some regenerants had several eyes joined
with pigment cells. Their eyes had been
arranged into two ,three or four eyes.
Supplementary eyes may come from defect in
regeneration process. Stability of eye
structure may demand functional interrela-
tion between eyes (optic nerves) and brain.
MYOSIN HEAVY CHAIN IN PLANARIAN
Kobayashi,S.,A.Yamada,H.Orii,K.Agata and
K.Watanabe. Lab.Regeneration Biol.,Fac.Sci
Himeji Inst. Tech., Harima Sci. Park City,
Akou, Hyougo, Dept.Biol., Fac.Sci.,
Hirosaki Univ., Hirosaki, Aomori.
Identification of molecules specific for
different types of cells is a key point on
understanding of planarian regeneration.
Although we have made many trials, it is
very difficult to prepare cell type speci-
fic monoclonal antibodies(MAs) by standard
method. Almost all MAs recognized sugar
and were not suitable for gene cloning. To
avoid this,we prepared polyclonal antiserum
against planarian tissue and screened an
expression library. Positive clones can be
used for analyzing the genes and also for
epitope selection,preparing the monospeci-
fic antibodies good for Western blot and
immunostaining. We report here structure
of a myosin heavy chain gene isolated using
this method. The myosin gene is similar to
that of mammalian muscle and exp-
ressed.in pharynx of planarian.
HOMEOBOX GENES IN PLANARIAN.
Orii,H., K.Agata and K.Watanabe
Lab.of Regeneration Biol., Fac.,of Sci.,
Himeji Inst. Tech., Harima Sci. Park City,
Akou-gun, Hyougo.
How does planarian regeneration occur?
Our knowledge of it is very poor, especial-
ly at the molecular level.
Homeobox genes have been isolated or identi
tified in many organisms belonging to di-
fferent phylogenetic groups and they
maybe play key roles in cellular differ-
rentiation and development. In order to
understand planarian regeneration, we
focused on planarian homeobox genes.
Using PCR technology and sequence analysis
we have identified 19 types of homeobox
genes in thts organism. This suggests
that planarian has a large number of
homeobox genes like nematode. We found
Antp-like homeobox gene in them, suggest-
ing that HOM-C gene cluster may be con-
served in planarian.
STUDY ON THE EYE-INDUCTIVE SUBSTANCE IN
FRESHWATER PLANARIANS.
S.Ishida and S.Matsuguti
Dept. of Biol., Fac. of Sci., Hirosaki
Univ., Hirosaki.
Though Lender called the eye-inductive
substance "organishine"(1952,1956), the
chemical nature has not been well known
yet. This time the next was clarified.
The substance is distributing over the
whole body, but it is activated only in
the brain. The inductive ability is
appeared beyond the concentration of 25
ug protein/worm in head extract of
Polycelis auriculata. It is supposed
that the substance is protein or
peptide. As the eyes are inducted by
the ammonium chloride treatment, it is
suggested that ammonium ion is one of
the materiales which active
eye-inductive substance. An inhibitory
substance against eye induction is
existing in the region which can not
regenerate their head in Bdellocephala
brunnea. There is no difference between
normal eyes and inducted eyes in
ultrastructure. it is possible that
ammonia excreted inside worm body is
one of the factor which act on the
increase of eye-number in Genus
Polycelis.
Developmental Biology 1187
DETECTION OF THE PLANARIAN (DUGESTA
JAPONICA) REGENERATION REGULATOR
S. Shiozaki, H. Fujino, T. Tanaka, M.
Ezaki, T. Saheki and T. Shinozawa. Dept.
of Biol. and Chem. Engineering, Faculty
of Engineering, Gunma Univ., Gunma
Lender described that crude homogenate
of planarian heads inhibited brain regen-
eration of decapitated planarians. In
this study, planarian head extracts were
fractionated by DEAE-Sephadex, CM-Sepha-
dex, Con A-Sepharose, Ammonium sulfate
precipitation, and Phenyl-Sepharose.
Each of the fractions were examined for
their effect on the growth of Neuro2a or
PC-12 cells. The 40% ammonium sulphate
precipitated fraction of the planarian-
head extracts inhibited the growth of
Neuro2a cells. More over, the same frac-
tion inhibited the brain regeneration of
decapitated planarians. These results
suggest that planarian head extracts
contain growth inhibitor of neuronal
cells and the utility of Neuro2a growth
assay in the purification of planarian
brain regeneration inhibitor.
References:
1. TH. Lender, J. Embryol. exp. Morph.,
8(3) 291-301, 1960
2. H. Fujino, T. Tanaka and T. Shinozawa,
Zool. Sci., 8(6) 1072, 1991
ENDOGENOUS ION CURRENTS AND REGENERATION
OF TAILS IN THE JAPANESE NEWT, CYNOPS
PYRRHOGASTER.
T.Nawata, College of Medical Sciences,
Tohoku Univ., Sendai.
Endogenous ion currents near amputated
newt tails were measured with a vibrating
electrode technique. The currents around
an intact tail before amputation were
less than 0.4yA.cm~ in density and
entered the surface of the tail. After
amputation of the tail, steady intens
currents with a density of 50-450yA.cm~
left from the end of the stump. The
stump currents continued to flow during
the first 2-3 days and declined to about
10A.cm~2 by 4 days after amputation. A
sign of regeneration of amputated tails
could be observed about 7 days after
amputation.
To maintain the intense stump currents
measured after amputation, an electric
current with an intensity of 0O.1yA was
artificially imposed on the end of the
stump over 14 days. Under this experi-
mental condition, a sign of regeneration
of amputated tails could be observed
about 3 days after amputation. In con-
trast, when such a current was imposed in
the reverse direction, the obvious regen-
eration was undetectable within 14 days.
These findings suggest that the en-
dogenous stump currents play some causal
role in initiating regeneration.
SUBCELLULAR LOCALIZATION AND MOLECULAR NA-
TURE OF THE no. 48 ANTIGEN FOUND IN XENOPUS
GERM PLASM BY FLUORESGENCE MICROSCOPY.
Ikenishi , K. Itoh” and M. Furusawa‘’.
Dept. of Biol., Fac. of Sci., Osaka City
Univ., Osaka and ~Furusawa MorphoGene Pro-
ject, ERATO, JRDC, Tsukuba. —
Subcellular localization and molecular
nature of the corresponding antigen to the
no. 48 antibody, which had strongly reacted
with the germ plasm (GP) or cytoplasm of
the germ line cells by fluorescence micro-
scopy in the previous study, were investi-
gated in Xenopus embryos.
The antigen was demonstrated to be pre-
sent on the GP-specific germinal granules
or their derivatives, mitochondria not only
in the GP or the cytoplasm of the germ line
cells but also in cytoplasm of every soma-
tic cells, and myofibrils of muscle cells
by immuno-gold labelling. In order to know
the molecular nature of the antigens on
those organelles, immunoblottings for the
subcellular fractions of the cleaving em-
bryos and the myofibrillar fraction of the
tadpoles were carried out. The Qo. 48
antibody recognized a Mr 53 x 10° band in
the precipitate of 7,000 g §"mitochondrial
fraction") and a Mr 40 x 10> band in the
supernatant of 20,000 g, but it did not
react with any band in the precipitate of
20,000 g (a "post-mitochondrial fraction"
in which the germinal granules are to be
contained). It also detected a Mr 43 x 10
band, probably actin, of the myofibrillar
fraction.
3
LIPID COMPOSITION IN VEGETAL, ANIMAL,
VENTRAL AND DORSAL HALF EMBRYOS DURING
EARLY DEVELOPMENT OF RANA JAPONICA.
M. Ryuzaki! and Y. Sone?. Dept. of 'Biol. and ?Biochem,
Kitasato Univ. School of Medicine, Sagamihara :
The authors conducted quantitative and qualitative
analysis of triglyceride (TG), phospholipid (PL) and total
cholesterol (TC) from vegetal(VE) and animal half
embryos(AE) at stages], 2 and 10, from vegetal 4-cells
and animal 4-cells at stage 5, and ventral (VnE) and
dorsal half embryos (DE) that divided near primitive gut
at stages 17 and 20 of Rana japonica. Weight percentages
of TG, PLand TC to total lipid content in these specimens
and their whole embryos were approx. 48-60% 15-27%
and 4-6% respectively. TG (52-53%, PL(21% and TC6%
in AE and TG(4485-50%, PL(27% and TCS-6% in VE
were the same for stage 1 and 2. TG and PL in VE and
VnE remaind unchanged following fertilization during
embryogenesis and throught stage 20, hatching, at ca 46-
50 %and 20-27% and in AEand DEat ca 53-60% and
15-21% respectively. Except for minor quantitatve
changes, the gross patterns of TG.PL and TC in the
spcimens showed no _ significant change during
embryogenesis. An anthron sulfuric acid positive lipid
was detected in a fertilized egg for the first time as a
single band and was also found in embryos at stagesS
and 10 during early development on TLC. But this band
was not detected in embryos at stage 17 or 20.
1188 Developmental Biology
SPATIAL DISTRIBUTION OF MITOCHONDRIAL
IrRNA IN DROSOPHILA EARLY EMBRYOS
S. Kobayashi, R. Amikura and M. Okada. Inst. of
Biological Sciences, Univ. of Tsukuba, Tsukuba,
Ibaraki
Polar plasm localized in the posterior polar
region of early Drosophila embryos contains
factors required for germ-line establishment. We
have reported that mitochondrial large
ribosomal RNA (mtIrRNA) is a cytoplasmic factor
that rescues embryos from u.v.-caused inability
to form pole cells, or germ-line cells. In this study,
we analysed spatial distribution of mtIrRNA in
early embryos using in situ hybridization
technique. MilrRNA was enriched in polar plasm
of cleavage embryos, but was no longer
enriched in pole cells. U.v.-irradiation to the
posterior polar region apparently lessened the
mtIrRNA signal in polar plasm. Furthermore,
Bic-D mutation, which mislocalizes the genetic
information for abdomen formation, but not one
for pole cell formation, in the anterior pole
region, never affected the localization of
mtIrRNA. These results suggest the possibility
that the enrichment of mtIrRNA in polar plasm
represents an indispensable step for pole cell
formation in normal development.
CELL DETERMINANT, MITOCHONDRIAL
LARGE RIBOSOMAL RNA, IN DROSOPHILA
CYTOPLASMIC DISTRIBUTION OF A POLE
EMBRYOS.
R. Amikura, S. Kobayashi and M, Okada. Inst.
Biol. Sci., Univ. of Tsukuba, Tsukuba.
Mitochondrial large ribosomal RNA
(mtIrRNA) is a candidate for the germ cell
determinants in the Drosophila embryo. We have
presented the idea that mtIrRNA is transported
out of mitochondria into the cytosol and takes
part in pole cell formation (Kobayashi and
Okada, 1989). In this study, we visualize this
phenomenon using the jn situ hybridization
method in electron microscopy.
We could detect mtIrRNA outside
mitochondria but not inside with this method. In
the early cleavage stage, mtIrRNA signal was
frequently found to attach to mitochondria. Some
of the signal was located on polar granules. On
the contrary, the control RNA signal (ND-1 RNA,
Mitochondrial small rRNA) was not detected from
cytoplasm nor from mitochondria. In the pole bud
stage, most of polar granules were free from the
signal. In pole cells, neither polar granules nor
nuclear bodies exhibited signal.
Considering those polar granules probably
contain pole cell determinants, the localization of
mtIrRNA in polar granules suggests the
importance of mtirRNA in pole cell formation.
CELL SHAPE CHANGES DURING DROSOPHILA
VENTRAL FURROW FORMATION.
M. Matsuda! and M. Yasutomi2. lDept. of
Embryo., Inst. for Dev. Res., Kasugai,
Aichi, “Lab. of Biol., Aichi Med. College,
Nagakute, Aichi.
Cell shape changes during ventral furrow
formation were measured by using NIH Image
1.41 in cross sections of Drosophila
embryos. To ascertain which changes are
responsible for the process, models of
ventral furrow formation were made. The
results indicated that changes of the
apical and basal cell surface width in the
presumptive mesoderm cells, which were
subdivided into the central and peripheral
cells, generated ventral furrow formation.
Apical cell surface elongation and basal
cell surface contraction in the peripheral
cells and the reverse phenomenon in the
central cells resulted in invagination of
the presumptive mesoderm cells. The
furrow deepened by the events that cells
located in the center of the central
population elongated their basal cell
surface and cells located in the side of
the central population elongated their
apical cell surface and contracted their
basal cell surface. The results also
indicated that the distribution of
microfilaments and the position of nuclei
May Correlate to changes of the apical and
basal cell surface width.
STUDIES ON A NEW MUTANT OF DROSOPHILA THAT
CHANGES DEVELOPMENT OF IMAGINAL DISCS. M.
Sawa’, M. Onuma?, M. Hatsumi*, K. I.
Wakahama* and Y. Oguma*. ' Dept. Biol.
Aichi Univ. Educ., Kariya, ? Dept. Biol.
Fac. Sci., Shimane Univ., Matsue, * Inst.
Biol. Sci., Univ. Tsukuba, Tsukuba.
We got a P[lwB) insertion strain, 416,
of Drosophila melanogaster that shows aber-
rations in adult morphology, degenerated
eyes, wings, legs, halteres, genitalia, and
testes in varing degree. Femurs, tibiae and
tarsi are degenerated or fused together.
Wings and halteres are severely reduced.
Testes are not elongated but spermatogesis
did not appear abnormal. The imaginal discs
of legs, wings and halteres from third
instar larvae were observed. The discs
derived from 416 lacked central regions
which correspond with distal part of each
organ in its fate map. The location of the
P element insertion of this strain was
mapped at 66B on the third chromosome by in
situ hybridization. Thirty eight excision
strains were constructed and twenty two
of them produced flies of normal phenotype,
which confirmed the aberrations in 416
should be resulted by the P[JwBJ insertion.
Ten strains showed mild defects and one
strain showed defects as severe as 416.
These may have deletions of DNA segments
which removed by the P element. Futhermore,
five excision strains were lethal, suggest—
ing that the product of the locus may be
indispensable for normal development.
Developmental Biology 1189
Sequential Appearance of Sensory Mother Cells
(SMCs) of Microchaetes on the Drosophila Notum:
Spacing Pattern of SMCs.
K. Usui, K.-I. Kimura. Laboratory of Biology,
Hokkaido University of Education, Iwamizawa
Campus, Iwamizawa, Hokkaido.
We have examined the spatio-temporal
appearance of SMCs of microchaetes during pupal
period using an enhancer trap line Al01 where lacZ
is expressed in SMCs. In the dorsocentral (DC)
region, microchaetes occur in evenly-spaced manner
in five longitudinal lines (L1-5 medio-laterally). At 8
hr after puparium formation (APF), SMCs were first
detectable in a DC line (L5) which runs anteriorly
through anterior and posterior DC macrochaetes. At
10 hr APF, two more lines of SMCs (L1&3) were
added in an even-spaced manner, in addition to LS.
Two hours later, moreover, two lines (L2&4) were
intercalated among these three pre-existing lines. In
each line, SMCs did not appear synchronously but in
a skipped pattern. In the case of the well-fed flies,
additional SMCs appeared in the largest space among
the five pre-existing lines of SMCs.
scute (sc) gene was ubiquitously expressed by
heat-shocking an hsp-sc chimeric gene in achaete/sc
mutant flies which lack all microchaetes. LS was
preferentially rescued in a stage-independent manner
during sensitive period, showing that the enhanced
sensitivity for SMC development existed in L5.
ROLE OF HEMOLYMPH 350kDa LECTIN ON PROGRA-
MMED CELL DEATH OF THE ANTERIOR SILK
GLANDS OF THE SILKWORM, BOMBYX MORI.
oie Soma »8: Sakurai-, K. Amanai* and T.
Ohtaki-. ept. Biolgy, Kanazawa Univ.,
Kanazawa, ,“Naatl. Inst. for Basic Biol.,
Okazaki, 3Nishogakusha Univ., Kashiwa,
Japan.
Silk gland, a larval specific tissue,
initiates to degenerate immediately before
pupation in the silkworm, Bombyx mori. At
the same time, the contents of 350 kDa
lectin is found abundantly in hemolymph.
Since hemolymph lectin was indicated to be
involved in non-self recognition in
Sarcophaga larvae, it was possible that
the Bombyx hemolymph lectin is involved in
the cell death of the silk gland,
especially in the removal of the gland by
phagocytes. Degeneration of the silk gland
is induced in vitro when the glands are
cultured with 20-hydroxyecdysone and
hemocytes. We examined the above
possibility by addition of gulucuronic or
galacturonic acid, both of which inhibits
hemagglutinating activity of the lectin,
or by adding an anti-lectin monoclonal
antibody to the culture to inhibit the
function of lectin. Under these
conditions, the gland degenerated,
indicating that the hemolymph 350 kDa
lectin may not be involved in the
degeneration of anterior silk gland.
CELL DEATH IN WING MORPHOGENESIS OF
LEPIDOPTERAN INSECTS 2
A.Yoshidg 1 R-Kodama , G.Eguchi™ and
T.Mitsui~. Project Office, Biohistry
Res. Hall, Tokyo, Div. of Morph.,, Natl.
Inst. Basic Biol., Okazaki, and ~Lab. of
Insect Toxicol. and Physiol., Inst. of
Phys. and Chem. Res. (Riken), Wako.
Degeneration of the marginal region in
the pupal wing epidermis is involved in
wing morphogenesis in the lepidopteran
insects (SUffert, 1929). First, we exam-
ined the pupal wing epidermis of the small
white cabbage butterfly, Pieris rapae,
with a light and an electron microscope.
Second, we injected BrdU into early pupae,
fixed them after 2h., and then detected
BrdU incorporated into the nuclei with
enzyme antibody technique.
Until 3rd day after pupation, morpho-
logical change do not occur in the degen-
eration region. The epidermis bulges out
dorsoventrally at 3.5th day, dramatically
contracts at 4th day, and finally disap-
pears at 4.5th day. At 3.5th and 4th day,
degenerating cells are visible along the
basal surface of the wing epidermis.
During 1 day after pupation, BrdU-
incorporating nuclei are visible in the
degeneration region, but almost absent
after 1 day. No mitosis is observed in
the degeneration region after pupation.
This suggests that cell death occurs after
DNA synthesis as in the digit formation of
the chicken embryo (Tone et al., 1988).
SPUR PATTERNS ON THE TIBIA AND FEMUR OF THE
GERMAN COCKROACH.
A. Tanaka and S. Kitamura. Dept. of Biol.,
Fac. of Sci., Nara Women's Univ., Nara.
Spur patterns were observed on the
tibias and femurs of all the legs
throughout postembryonic development of the
German cockroach, Blattella germanica (L.).
All the spurs were named by a row-and-level
method. Six rows of spurs were recogniz-
able on the tibia in the adult stage. One
of the rows on the posterolateral surface
was designated as row 1. Five remaining
rowS were named consecutively as rows 2-6,
clockwise or anticlockwise over the distal
face of the right or left tibia,
respectively. Spurs belonging to the same
row were named alphabetically from the
distal level. The number of spurs per row
differed with rows and on the three pairs
of legs. A clear gradient was found with
respect to the size of spur: the more
distal ones in the same row tended to be
larger than the more proximal ones. Three
rowS were recognizable on the femur and
designated as rows I-III. Levels of spurs
were also named alphabetically from the
distal position. Spurs on the femur were
not always larger more distally but rather
showed double gradients with respect to
size. In the larval stage, both on the
tibia and on the femur, spinelike hairs
replaced spurs at some positions of the
rows and levels. The number of spurs
steadily increased instar by instar until
the last instar.
1190 Developmental Biology
ON THE OOGENESIS OF FRESH WATER HYDRA AND SOME
SPECIES OF MARINE HYDROZOA
K. Noda!, C. Kanai!, S. Kubota® and H.Namikawa%
1Ultrast.Res. Tokyo Metropol. Inst. of Gerontol.
Tokyo, @Seto Marine Biol. Lab., Kyoto Univ.
Shirahama. “Dept. of Zool.,Natl.Sci.Museum. Tokyo
Many oocyte-groups appeared among numerous oo-
gonia in an egg-forming area of fresh water
polyp, Pelmatohydra robusta. But, only one cell
of the oocytes situated at the central part of
an egg-forming area was always preferred as pre-
sumptive egg by ectodermal epithelial cells
After one oocyte was selected, the base of ecto-
dermal epithelial cells of egg-forming area were
broken. We also examind a course of oogenesis
during morphogenesis of gonophore/medusa of 7
species of marine hydrozoa in special refer-
ences to 1) the stage of germ cells migrating
into inner ectoderm, 2) migrating route of germ
cells, 3) the number of oocytes in an egg-
forming area and 4) structures of gonophore/
medusa when germ cells developed to oocytes (in
inner ectoderm). Marine materials used in the
study were anthomedusa, Eudendrium, 3 species
of Stylactaria, Halocordyle disticha, Sarsia
japonica, and leptomedusa, Eugymnathea japonica
Final structure of gonophore/medusa seemed to
be closely depend on the pattern of oogenesis
exhibited by each species.
REGURATIVE DEVELOPMENT OF HYDRA EMBRYO
T. Oda
Dept. of Biol., Fac. of General Edu.,
Yamagata Univ., Yamagata.
Hydra embryos in various stages were divided
with razors edge and the subsequent development
of their isolated blastomeres or halves were ob-
served. When the blastomeres from 2- and 4-cell
embryos were separated, some of the cells develop-
ed into a complete and well-proportioned adult.
Thus, the early cleavage stage blastomeres could
regulate their development so as to produce a com-
plete organism. When embryos at the hollow blas-
tula and gastrula stages were cut in half meridi-
onally through animal and vegetal poles, both
halves produced entire organisms. Moreover, when
embryos at the same stages were split equatorially
separating animal and vegetal poles, both halves
also developed into normal organisms. These may
happen because the cells of early embryo are equi-
potential in the sense that there is no difinite
relation between the position of any of the blas-
tula cells and the specific tissue it will form in
the developing embryo. This idea may be supported
by the following observation. Gastrulation in
hydra was caused by the multipolar ingrowth, in
which endodermal cells migrated into blastocoel
from not only vegetal pole but also other posi~
tions of ectodermal layer. From the results, it
became clear that hydra embryo from 2-cell to gas-
trula stage had a great ability to regulate devel-
opment.
BASAL DISK FORMATION IN HYDRA ;PROSPERITY
AND DECAY OF RF-AMIDE+ NEURONS DURING THE
FOOT REGENERATION
¥..KOBAYAKAWA® A.MAKI2 & O.KOIZUMI?
Ol. ibe) 5 Coll. gf Genl. Educ., Kyushu
Univ., Fukuoka, ‘Physiol. Lab., Dept.
Sci., Fukuoka Women’s Univ., Fukuoka
RF-amide+ neurons are located in the
tentacles, hypostome and peduncle, but not
in the body column or basal disk in Hydra
magnipapillata. Ectodermal epithelial
cells also show position specific charac—
ters. In the basal disk, epithelial cells
are special mucous secreting cells (BDGCs
;Basal Disk Gland Cells).
When a hydra was amputated its foot
at the middle part of the body column, as
the first step of foot regeneration, the
epithelial cells at the proximal end
differentiated BDGCs. Then, the RF-amide+
neurons reappeared in the new peduncle
region, but not in the basal disk region.
When a hydra was amputated its basal
disk at the lowest part of its peduncle,
RF-amide+ neurons remained at its proximal
end. Then, the epithelial cells at the
proximal end began to differentiate BDGCs.
The RF-amide+ neurons, however, did not
begin to decay from the proximal end
before the basal disk regeneration pro-
ceeded to a certain extent.
It is suggested that during the foot
regeneration in hydra, the epithelial cell
differentiation precede the change of
neuropeptide phenotype in nerve net.
NERVE NET FORMATION BY NERVE PRECURSOR
(INTERSTITIAL) CELLS INTRODUCED INTO NERVE-
FREE HYDRA
S.Minobe and O.Koizumi.
Physiol.Lab.,Fukuoka Women’s Univ., Fukuoka
Hydra has a simple nervous system
consisting of a nerve net that extends
through the body. The nerve cells are
continuously produced by the
differentiation from interstitial
multipotent stem cells even in adult hydra.
Hydra consists of two independent cell
lineages, epithelial and interstitial cell
lineages.
In this study, epithelial hydra that
contains no nerve cells was produced.
Hydra was treated with colchicine to
eliminate interstitial cells. The resultant
hydra could be maintained by force-feeding.
Nerve net formation was examined after the
interstitial cells were introduced into the
epithelial hydra.
The nerve net of both ganglion and sensory
cells had formed from the base of tentacles
toward the tip. The rate of the nerve net
formation corresponded with that of the
displacement of the tentacle epithelial
cells. Unlike neurons, nematocystes had
recovered quickly through the tentacles.
Thus the mechanisms for the repopulation
are different between nerve cells and
nematocytes. In conclusion, the nerve net
formation occurs at the base of the
tentacles and then it is displaced along
the tentacles with the epithelial tissue.
Developmental Biology 1191
REGENERATION PROCESS IN HYDRA CELL AGGRE-
GATES ANALYZED BY THEIR DEVELOPMENT OF
ORGANIZED MOTION.
Sato-Maeda,M.*,and Tashiro,H.,Photodynamics
Res Ctr, Inst Phys/Chem Res (RIKEN) ,Sendai.
i Hydra can regenerate even from a disso-
ciated cell aggregate. To know how and when
an aggregated medley of dissociated cells
initiate self-organization to form the body
axis which may guide the direction of the
subsequent growth, we analyzed motion of ag-
gregates in early stages of regeneration.
Aggregates made of cells which are selec-—
tively gathered from the gastric region of
intact animals were prepared in the size in
which only one head was expected to form.
After the final dilution of medium at t=
22hr, the motion of an aggregate was con-
tinuously recorded by a timelapse VIR. Both
the widths of x- and y-axis directions of
the aggregate during the period were mea-
sured with an image processor. Until 30hr,
aggregates stayed spherical. They showed
slow expansion or slow asymmetric motion.
Aggregates started to show distinct
anisotropic motion with a single axis after
a few times of big contractions. The ampli-
tude and frequency of the asymmetric motion
increased. Taking account of observations
for tissue-piece or aggregates of low nerve-—
density, these results are understood that
(1)the determination of the body axis was
correlated to the appearance of the an-
isotropic movement and (2)the nerve system
that is completed later relates twitching of
spike-like contraction-recover motion.
A NERVE DERIVED FACTOR REQUIRED FOR SUR-
VIVAL AND PROLIFERATION OF INTERSTITIAL
STEM CELLS IN HYDRA
T. Fujisawa. Natl. Inst. of Genetics,
Mishima
Interstitial stem cells (i-cells) in
Hydra proliferate continuously in asexual-
ly budding animals, but their density is
always maintained at a constant level in
the tissue.
In the course of the study how this
i-cell homeostasis is maintained, I have
found that some factor(s) derived from
nerve cells is required for survival and
proliferation of i-cells. Partial purifi-
Cation and characterization of the factor
Suggest that it is a neuropeptide which
resembles head activator (Schaller and
Bodenmueller, 1981). Synthesized head
activator in a dimerized form (a gift from
Dr. Schaller) also exhibited an activity
to support survival and proliferation of
i-cells.
These results suggest that head
activator is at least one of the paracrine
growth factors of i-cells in Hydra.
A SUBPOPULATION OF INTERSTITIAL CELLS IN
HYDRA MAGNIPAPILLATA THAT IS RESTRICTED
TO DIFFERENTIATE INTO GERM CELLS.
C. Fujisawa! and T. Sugiyama?. !Dept. of
Genetics, School of Life Science, Grad. Univ. for
Advanced Studies, Mishima and 2Natl. Inst. of
Genetics, Mishima
Interstitial cells (i-cells) in hydra are
undifferentiated stem cells that proliferate
continuously. They also differentiate into nerve
cells, nematocytes (stinging cells), gland cells
and germ cells (eggs and sperm).
To examine the differentiation capacity of
individual i-cells, we used hydroxyurea
treatment to obtain i-cell clones from a male
strain of Hydra magnipapillata. We have
previously cloned i-cells which differentiated
only into sperm (1991 Meeting of Japan Soc. of
Devl. Biologists). Using the same strain (male),
we have also isolated, to our surprise, i-cell
clones which differentiate only into eggs.
These results suggest that male polyps
contain a subpopulation of i-cells restricted to
egg differentiation, whose differentiation is
Suppressed in male tissue.
THE DIGESTIVE TRACT OF THE ASCIDIAN
POLYANDROCARPA MISAKIENSIS. |. ESOPHAGUS.
H.Koyama, Dept. Anat., Sch. Med., Yokohama
City University, Yokohama.
The organization of the esophagus and
its associated structures was studied ina
styelid ascidian, Polyandrocarpa
misakiensis. The simple squamous branchial
epithelium gradually becomes thick and
possesses apical metachromatic granules at
the entrance to the esophagus. The
esophagus is surrounded by connective
tissue, in which many blood sinuses and
terminal branches of the pyloric gland are
embedded. The peribranchial epithelium
Surrounds the esophagus as a rough
periesophageal epithelium containing many
coelomic cells. Many circular and
longitudinal smooth muscles are present
between the pene soplegeas. epithelium and
esophagus. The lumen of the relaxed
esophagus has three (left, right, and
posterior) main folds. Pseudostratified
regions occur at the base of each fold.
The remaining part of the esophagus is a
simple columnar epithelium, which mainly
consists of ciliated mucous cells. The
anterior basal side of the left fold has a
group of cells different from typical
mucous cells. The mucous cells have some
Cilia, microvilli, granules, much rER,
Golgi apparatuses, and mitochondria.
There are two types of granules, one large
and electron-translucent, and the other
small and electron-dense. These granules
lack a limiting membrane. There are also
paraneuron-like cells with apical
microvilli and dense cored vesicles mostly
in their subnuclear regions.
1192 Developmental Biology
ON THE EGG CYTOPLASMIC FACTORS RESPONSIBLE
FOR THE MUSCLE CELL DIFFERENTIATION OF
ASCIDIAN CIONA SAVIGNYI: EGG FRAGMENTS
PRODUCED BY CENTRIFUGAL FORCE.
Y. Marikawa and N. Satoh. Dept. of
Zoology, Fac. of Sci., Kyoto Univ., Kyoto.
Dechorionated unfertilized eggs of C.
Savignyi were incubated for 10 min in the
sea water that contains 10 ug/ml
cytochalasin B and then centrifuged for 20
min at 1500xg. ‘These treatments separated
eggs into several types of fragments,
large red fragments (-~60% egg volume),
small gray ones (~10% egg volume), and
small transparent ones. Red fragments
contained egg chromosomes. If the
fragments were inseminated, they developed
into the so-called permanent blastulae, in
which epidermal differentiation was
evident but muscle cell differentiation
was not detected. Gray and transparent
fragments never cleaved even if they were
inseminated. However, if red fragments
were inseminated after the fusion with
gray fragments they developed into the
embryos in which muscle cell
differentiation was conspicuous. The
embryos derived from these fused fragments
sometimes appeared as normal tadpole
larvae. These results suggest that muscle
determinant in the eggs was mainly
Partitioned into gray fragments and not
into red fragments.
AN ATTEMPT TO ISOLATE cDNA CLONES
SPECIFIC FOR B4.1 BLASTOMERES OF THE
ASCIDIAN EMBRYO
T. Miya!, K. W. Makabe, 2 and N. Satoh!
1Dept. of Zool. , Fac. of Sci. , Kyoto Univ. , Kyoto ,
2Div. of Biol. , Califommia Inst. of Tech. , Pasadena , USA
During ascidian embryo genesis, differentiation of primary
lineage muscle cells, which are derived from B4.1
blastomeres of the 8-cell embryo, seems to be controlled by
factors localized in the egg cytoplasm. To analyze mRNAs
specific for B4.1 blastomeres, we collected B4.1
blastomeres and blastomeres of the animal hemisphere
(a4.2+b4.2) from 8-cell stage embryos of the ascidian
Halocynthia roretzi, and we made each cDNA library.
Then we made a subtracted cDNA library, and by
differential screening we isolated some cDNA clones which
are present in the B4.1-cDNA library but not in the animal
hemisphere-cDNA library. Now, we are continuing farther
analysis of these clones.
LOCALIZATION PATTERNS OF CYTOSKELETAL
COMPONENTS AND MYOPLASMIN-C1 DURING EARLY
DEVELOPMENT OF THE ASCIDIAN EMBRYO.
Miki Y., Ashida K., Tanaka R., Yokoyama K.
and Nishikata T. Dept. of Biology, Fac. of
Science, Konan Univ., Kobe.
The myoplasm of the ascidian egg is believed to
contain muscle determinants, and is distributed
into muscle precursor blastomeres through the
ooplasmic segregation. The segregation consists
of two phases, each mediated by different systems,
the first by microfilaments and the second by
microtubles (Sawada & Schatten, 1989).
Myoplasmin-Cl is one of the myoplasmic
components which are thought to play an important
role in the muscle cell differentiation. In order
to examine the interaction of myoplasmin-Cl and
other cytoskeletal proteins, we immunologically
detected tubulin, actin and p58 (resembles to the
porcine neurofilament 160; Swalla et al, 1991)
with myoplasmin-Cl on the same sections. During
the first phase of the segregation, the
distribution of myoplasmin-Cl was closely related
to that of the actin rather than the tubulin. On
the second phase, myoplasmin-Cl migrated
posteriorly together with the sperm aster. As far
as we examined, myoplasmin-Cl and p58 segregated
to the same area of the egg during ooplasmic
segregation. These results implicate the image of
the myoplasm which is composed of the intricate
complex of the cytoskeletal components; these
components dynamically move, rise and fall,
associate and disassociate with each other.
ANALYSIS OF THE EXPRESSION OF THE MYOGENIC bHLH
PROTEIN GENE IN THE ASCIDIAN 4alocynthia roretz2.
I.Araki!, N.Satoh!, K.W.Makabe?, H.Saiga®>.
Dept. of Zool., Fac. of Sci., Kyoto Unaiv.,
Kyoto, *California Institute of Technology,
Pasadena, *Dept. of Biol., Fac. of Sei.,
Tokyo Metropolitan Univ., Hachi-oji.
To address the question whether MyoDi-like
factors play pivotal roles in muscle
differentiation in #4.roretzz embryo, we isolated by
PCR method two cDNA clones and one genomic clone
encoding a protein which belongs to the myogenic
bHLH protein family. The difference hetween cDNA
clones occur in the poly(A) sites. The gene
consists of four exons. In the 5’ upstream region
four’CANNTG motifs (or E-boxes) are clusterd around
-550. They may be target sites of its
autoregulation. This protein consists of 435 amino
acid residues and its bHLH domain is well
conserved.
RT-PCR analysis has demonstrated that the
expression of this gene begins at 64-cell stage and
continues until swimming larva stage. In adult,
body wall muscle and heart express this gene, while
liver, gill and intestine don't, although none of
the members of the myogenic bHLH protein gene was
expressed in the heart of mammals. These results
suggest that a MyoDl-like factor may be involved in
ascidian muscle differentiation
Developmental
ACTIVITY OF THE UPSTREAM REGION OF
HALOCYNTHIA RORETZI MUSCLE ACTIN GENE IN
CIONA SAVIGNYI EMBRYOS.
A. Hikosaka, T. Kusakabe, K. W. Makabe,
and N. Satoh. Dept. of Zoology, Fac. of
Sci., Kyoto Univ., Kyoto.
HrMA4a, one of the muscle actin genes of
the ascidian H. roretzi, is expressed
specifically in larval muscle cells during
embryogenesis. We have already shown that
about 1.4-kb 5' upstream region of this
gene has the function to control muscle-
specific activation of this gene. In this
study we examined whether the upstream
regulatory region of HrMA4a works in the
other ascidian Ciona savignyi.
We constructed pHrMA4a-Z that contains
the upstream regulatory region of HrMA4a
and coding region of bacterial €-galacto-
sidase gene (lacZ). We injected this
fusion gene into C. savignyi eggs,
cultured them until hatching, and assayed
for B-galactosidase activity by
histochemical staining. It was observed
that the gene was expressed predominantly
in larval muscle cells. However, in some
cases the gene expression was detected in
unidentified cells located at the ventral
side of larval trunk region and in some
other cases it was also activated in
mesenchyme cells although its expression
was weak. pHrMA4a-Z deletion mutant that
has only 216 bp upstream region was also
expressed in basically same pattern,
although in some specimens it was
expressed in larval brain cells.
AN ATTEMPT TO ISOLATE MSH GENE OF THE
ASCIDIAN HAROCYNTHIA RORETZI
K.Tagawa and N.Satoh. Dept.Zool.,Fac.Sci.,Kyoto
Univ., Kyoto.
msh (muscle segment homeobox) is a gene that has been
isolated from Drosophila, and known to be expressed in the
mesoderm, developing CNS and the muscle of the fly. In
order to study whether msh gene homolog of H.rorerzi plays
an important role in the ascidian development, we tried to
isolate msh gene homolog of H.roretzi. We constructed a
genomic library of H.roretzi and screened the library using
Ciona intestinalis msh as a probe. So far, we obtained
several canditate clones, of which sequences are now
investigated.
Biology 1193
AN ATTEMPT TO ISOLATE A NOTOCHORD SPECIFIC
GENE OF THE ASCIDIAN HALOCYNTHIA RORETZI
H. Yasuo and N. Satoh. Dept. Zool. , Fac. Sci. , Kyoto
Univ. , Kyoto.
The ascidian is primitive chordate, which develops a
notochord only during larval stage. We want to know the
origin of notochord in the animal phylogeny, by studying of
the molecular mechanism of the notochord cell differentiation
in the ascidian.
The Brachyury (T) gene is a genc that has been
isolated in mouse and Xenopus , and the gene is specifically
expressed in their notochords. The amino acid sequences of
the putative Brachyury proteins of mouse and Xenopus are
highly conserved in the N-terminal half. By using the
Xenopus Brachyury cDNA fragment encoding the
conserved region as a probe, we isolated a genomic clone of
an ascidian homolog of Brachyury . We used this clone to
probe Northern blots of RNA prepared from H. roretzi
embryos at different stages. Maternal transcript was not
detected. A transcript, about 2.3kb in length, was first
detected in gastrula,and the expression was maximal at
gastrulation. The amount of the transcripts, however,
decreased following neurula stage. The size and the
expression pattern of the ascidian gene closely resemble
those of mouse and Xenopus Brachyury genes. Now we
are screening a gastrula cDNA library with a probe prepared
from the genomic clone.
THE MOLECULAR STRUCTURE OF cDNA CLONES
SPECIFIC FOR THE EPIDERMIS OF THE ASCIDIAN
EMBRYO.
1T.Ueki, 7K.W.Makabe and!N.Satoh. Dept. of Zool.,
Fac. of Sci., Kyoto Univ., Kyoto. 2Div. of Biol., California
Inst. Tech., Pasadena. USA.
To investigate the molecular mechanism involved in the
differentiation of epidermal cells of the ascidian embryo, we
have isolated eight different cDNAs for mRNAs that are
specifically expressed in the epidermal cells of Halocynthia
roretzi embryo. The temporal expression patterns of the
mRNAs have been revealed to be classified into four types.
We choosed four genes, HrEpiA,B,C and E, that represent each
of the four types. We isolated and analysed longer cDNA
clones of them. The sequences of the cDNAs were determined
by dideoxy method. The longest cDNA of HrEpiA was about
1.2kb, nearly full-length of its mRNA. It encodes a 241amino-
acid peptide started from the first methyonine and the peptide
sequence was revealed to be similar to epimerases. The longest
cDNAs for other genes,HrEpiB and C, were several hundred
bases shorter than the predicted size of their mRNAs. The
amino acid sequences predicted by the longest open reading
frame were used for the database search, but the search
revealed no significant similarity between the amino acid
sequences and any previously reported protein sequences. The
longest cDNAs for the HrEpiE gene so far obtained was about
1 kb shorter than its 3.0 kb mRNA. This cDNA was thought
to cover length of the trotein not long enough for database
search.
1194 Developmental Biology
Tissue compartments in Drosophila gut
epithelium: minimum tissue units of gene
expression.
R. Murakami, A. Shigenaga!, A, Matsumoto’,
I. Yamaoka and T. Tanimura’. Biol.
Inst., Yamaguchi Univ., Yamaguchi. Biol.
Lab., Kyushu Univ. Ropponmatsu, Fukuoka.
Spatial expression patterns of lacZ
reporter gene were analyzed in the gut of
Drosophila larvae transformed with P
elFement-lacZ vector (P-lacZ). lacZ
expression by this method has been shown
to reflect the expression of a gene
neighbouring the insertion of P-lacZ
vector. lacZ-positive cells formed
distinct domains with transverse and
longitudinal boundaries along the gut
tube. Boundaries of lacZ-positive
domains were included in consistent
demarcation lines which subdivided the gut
epithelium into about 40 partitions
regardless of the strain. We called the
partitions "tissue compartments". lacZ-
positive domains of the strains appeared
as a single tissue compartment or as a
combination of several compartments,
Overlapping with each other. All the
cells in a tissue compartments were
equivalent as to lacZ expressions,
suggesting that the tissue compartment is
a minimum tissue unit of gene expression
comprising the cells with a common gene
expression pattern.
Analysis of the proctodeum gene which
causes malformation of the hindgut of
Drosophila embryo.
A. Shigenaga, I. Yamaoka and R. Murakami.
Biol. Inst., Yamaguchi Univ., Yamaguchi.
A recessive lethal mutant with
malformation of the hindgut was isolated
from P-lacZ enhancer trap strains. We
called the mutant proctodeum (proc) since
the proctodeum (hindgut blastema)
degenerated during stage 12-14 in proc
embryo. Target tissue of this mutation
corresponded to JacZ-positive domain in
the proc embryo, suggesting that the lacZ
expression of this strain represented the
expression of the proc gene. lacZ
expression was first detected shortly
after the beginning of cellularization at
stage 5. lacZ-positive domain formed a
ring covering approx. 10-20 % egg length.
This domain included future ectodermal
proctodeum and visceral mesoderm of the
hindgut. lacZ activity continued to be
detected in the developing hindgut in
embryos of later stages. lacZ activity
was still positive in hindgut epithelium
of larva and adult. Since the lacZ of the
Proc was not expressed in the trunk and
torso embryos in which anteroposterior
axis was disorganized, expression of the
proc was thought to be under the control
of these maternal genes.
PRODUCTION OF A MONOCLONAL ANTIBODY
AGAINST KERATIN EXPRESSED IN THE CHICKEN
EMBRYONIC DIGESTIVE TRACT EPITHELIUM
K.Sato, K.Urase and S.Yasugi. Dept. Biol., Fac., of Sci.,
Tokyo Metropolitan Univ., Tokyo
We found that the expression of certain keratin molecules was
related to the differentiation of chicken embryonic digestive
tract epithelium. To investigate more precisely the nature and
changes of keratin molecules during development, we tried to
get monoclonal antibodies against chicken keratins with using
s-carboxymethylated extract of day 13 chicken gizzard (GZ) as
antigen.
We obtained a monoclonal antibody (IgM) which recognized
72 and 78.5 kDa polypeptides on immunoblotting after SDS-
PAGE of the extract. We examined the staining patterns by this
antibody in the whole embryos in early stages (day 1- 4) and
the digestive organs (from day 5) . In early stage embryos and
in digestive organs, the endoderm gave consistently positive
signals. In the GZ and proventriculus (PV), the positive
structures appeared mainly as filaments running parallel to the
apico-basal axis of the epithelial cells. In the GZ epithelium
from day 14 afterwards, the staining pattem changed from
filamentous to granular. In the PV, from day 8 afterwards when
the deep glands begin to be formed, the positive structures in
the cells of deep part of the glands became granular and were
more faintly stained. The outermost layers of epithelium of
ectodermal derivatives such as skin and scale also positively
reacted to the antibody.
SUPPRESSION OF DNA SYNTHESIS IN THE CHICKEN
EMBRYONIC DIGESTIVE ORGANS BY AN INHIBITOR OF
PROTEIN KINASE C.
Sadao Yasugi! and Etsuko Yasugi?. 1Dept. Biol.,
Fac. Sci., Tokyo Metropol. Univ., Tokyo, and 2Div.
Clinic. Biochem., Clinic. Inst., Natl. Med. Center,
Tokyo, Japan.
We reported the suppressive effect of H7, a potent
and specific inhibitor of protein kinase C (PKC), on
the differentiation of digestive organs of chicken
embryos in vitro. To analyze the _ relationship
between suppressive action of H7 and proliferation
of epithelial and mesenchymal cells of organs, we
cultivated 6-day proventriculus and intestine and
assayed the incorporation of BudR into DNA of cells
of these organs on the 3rd day of cultivation when
the suppression of epithelial differentiation was
already observed. In the case of proventriculus,
the control explants showed the labeling of 6.2% of
mesenchymal cells and 14.9% of epithelial cells.
When 100 uM of H7 was added to the culture
medium, the labeling index in mesenchyme lowered
to 2.3%, whereas that of epithelium remained at the
level comparable to that of control explants. The
coexistence of H7 and SC9, an activator of PKC, in
the medium restored the labeling index of mesen-
chyme to 4.7%. The similar tendency was observed
as to the proliferation of intestinal explants. These
data suggest that H7 affects primarily the prolifer-
ation rate of mesenchymal cells and brings about a
reduced growth, and this in turn results in the
suppression of normal epithelial differentiation.
Developmental Biology 1195
DIFFERENTIATION OF CHICK EMBRYONIC OESO-
PHAGEAL EPITHELIUM CULTURED IN A BASEMENT
MEMBRANE-LIKE MATRIX.
S.Matsushita. Dept.of Biol., Tokyo Women’s
Medical College, Tokyo.
The oesophageal epithelium of 6-day-old
chick embryos isolated by collagenase-
treatment was embedded in a small volume
of a basement membrane-like matrix
(Matrigel), placed at the interface of air
containing 5% co, and a liquid medium con-
sisting of 75% Medium 199, 20% 12-day
chick embryo extract and 5% foetal bovine
serum, and cultured at 38°C.
The oesophageal epithelium, which had
been pseudo-stratified columnar-type and
had a tubular shape, came to form a small
vesicle within one day in culture, and be-
came stratified-type within 2 days. After
4 days, part of the epithelium became
mucus-secretory type, while the rest of
the epithelium increased in cell layer and
developed cilia on the luminal surface.
After 6 days, mucous epithelium and strat-
ified squamous epithelium containing many
tonofilaments were found, which resembled
the glandular and luminal epithelia of the
mature oesophagus of hatched chicks, res-
pectively. Thus, the present culture meth-
od could support the full differentiation
of the oesophageal epithelium of the 6-day
chick embryo in the absence of mesenchyme.
THE INFLUENCE OF A NEW ANESTHETIC AGENT
‘ISOFLURANE ' ON THE HATCHED CHICKEN.
-Nonoyama , H.Tanaka and H.Haseggwa O
Aichi Univ. of Educ., Kariya and ~“Chukyo
Womens Univ., Ohbu.
The authors reported on the anesthetic
agents such as halothane, enflurane and
sevoflurane caused the inhibition of grow-
th,liver damage and malfolmation of chick
embryos previously. In this studies, we
examined on the influence of a new anesth-
etic agent, isoflurane to the hatched chi-
cken according to the previous reports.The
ratios of the body weights of 2% treated
chickens were suppressed 16.4% and 18.2%
on 20 and 25 days after respectively, as
compared with the control. The values of
serum GOT, GPT and r-GTP of the control
were 340, 7, 27; on the contrary the val-
ues of the treated chickens were 391, 7,
25 (after 20 days),and 439, 8, 21 (after
25) respectively. The variation of serum
LDH patterns ofthe control was as follows:
Ds YW = /Oo8, wo As 1760, wos 3S 2 5a,
LDH 4 = 2.1 and LDH 5 = 3.3. And that of
the 20 days' treated hatched chicken was
51.6, 15.8, 7.8, 8.2, and 16.6; and the
a5 Casyys Was @Soilp Wolh Vas, BoB, ~ evacl
18.8 respectively. Many stretched micro-
villis and lymphatic cells on the surface
of disse cavity of the treated chicken
liver were obserbed. However, not so sig-
nificant difference was recognized bet-
ween the treated chicken liver and the
control, from the scanning electron mic-
roscopic observation.
POSSIBLE INVOLVEMENT OF GP-100 GLYCOPROTEIN
IN THE PROCESS OF BUDDING AND REGENERATION
OF THE POLYSTYELID ASCIDIAN, POLYANDROCARPA
MISAKIENSIS.
Y.Katsuyama , S. Fujiwara and K.Kawamura .
Dept. of Biol. , Fac. of Sci. , Kochi Univ., Kochi
GP-100 is a glycoprotein that binds to the galactose-specific
lectin, T'C-14 extracted from the polystyelid ascidian
Polyandrocarpa misakiensis. Purified GP-100 was separated
into two subunits (20kDa and 26kDa) on SDS-PAGE under
reduced condition. In the asexual life span, it increased remarkably
in amount during budding, and decreased to a minimum level in
functional animals of pre-budding stage. GP-100 also increased
in amount in the process of regeneration, although the amount
was not so high as that of developing buds. We prepared anti-
GP-100 monoclonal antibody that recognized specifically the
20kDa subunit of the glycoprotein. Immunostaining showed
that in adult animals GP-100 was localized in the mesenchymal
space between the epidermis and atrial epithelium, indicating
that this glycoprotein is a major coelomic component. GP-100
was also located in the vacuoles of a certain kind of blood cell,
probably leucocyte. Thus, this kind of cell is considered as
GP-100-producing cell. Interestingly, GP-100 disappeared from
the morphogenetic region of both developing buds and
regenerating adult animals.GP-100 was also extractable from
other budding polystyelid ascidians such as Metandrocarpa, but
not from solitary ascidians and budding entrogona such as
Aplidium. The results of this study suggest that GP-100 might
be involved in budding and regeneration of polystyelid ascidians,
but that it might act as a negative regulator of morphogenesis
rather than a factor that promotes morphogenesis.
Retinoic acid can induce the ectopic morphogenesis
via mesenchymal cells in the palleal bud of
Polyandrocarpa misakiensits. K.Hara, S. Fujiwara,
K. Kawamura, Dept. of Bio., Fac. of Sci., Kochi
Univ. Kochi.
We have already shown that retinoic acid (RA) can
induce the secondary antero-posterior axis in the
palleal bud of Polyandrocarpa misakiensis
In this study, we examined whether mes en-
chymal cells could mediate the effect of RA on the
induction of the ectopic morphogenesis. The mesen-
chymal cells were isolated from adult animals and
treated with 10°>M RA, or DMS Oas a control. They
were implanted into the bud to assess their morpho-
genesis inducing-activity. RA treated mesenchymal
cells could induce the ectopic morphogenesis in 45%
in the most efficient cases, but DMSO treated ones
could not do so. This activity of mesenchymal cells
depended on the time length of exposure to RA,
during which RA had no apparent effect of growth
of the mesenchymal cells. These results indicated
that RA could induce the ectopic morphogenesis via
the mesenchymal cells. They also suggested that the
mo rphogenesis-inducing activity of mesenchymal
cells was acquired without any cell growth.
1196 Developmental Biology
DEVELOPMENTAL ROLE OF ALDEHYDE
DEHYDROGENASE IN THE PROCESS OF
BUDDING AND REGENERATION OF THE
TUNICATE, POLYANDROCARPA MISAKIENSIS.
K. Kawamura and S. Fujiwara. Dept. of Biol., Fac.
of Sci., Kochi Univ., Kochi.
We have previously shown that in the budding
tunicate Polyandrocarpa misakiensis retinoic acid can
induce an ectopic morphallactic field of developing
buds. From this animal. we extracted endogenous
retinoids and, using HPLC, identified some major
peaks as cis-retinal, all-trans-retinal and all-trans-
retinoic acid, of which cis-retinal was most abundant.
During both budding and regeneration, cis-retinal
decreased remarkably in amount. Concomitantly,
aldehyde dehydrogenase that could metabolize retinal
in vitro was induced in epithelial cells and then in
mesenchymal cells. Both kinds of cells were major
sources of endogenous retinoids. In developing buds,
the enzyme always appeared at the proximal end. that
is, the future morphogenesis domain, whereas in
regenerating animals it was observable at the whole
area of the zooid piece. When exogenous retinoic acid
was applied to buds, the enzyme was induced in the
secondary morphallactic field, as in the primary field.
The results of this work show that Polyandrocarpa
aldehyde dehydrogenase is a potent retinoic acid
synthase induced specifically in the process of
budding and regeneration. They also suggest that the
enzyme plays a role in morphallactic development of
Polyandrocarpa buds and regenerates via positive
auto-regulation of retinoic acid synthesis.
THE INHIBITION OF SPERMATOGENESIS IN THE
MEDAKA, ORYZIAS LATIPES, EXPOSED TO HIGH
WATER TEMPERATURE.
S. Haniaeuicnet ; M. Takakuwa2
Yamazaki
Dept. Biol., Coll. Gen. Educ., “Dept.
Biol., Fac. Sci., Niigata Univ., Niigata
and
It is well known that spermatogenesis
is inhibited when mammalian testes are
exposed to high temperature. We reared
the medaka, Oryzias latipes, at 27, 30, 33
and 36°C of water temperature, and found
that the inhibition of spermatogenesis
occur in 33 and 36°C groups. From the
analysis of the areas occupied by germ
cells at each stage of spermatogenesis,
conspicuous decrease in the type B sperma-
togonia were noted in 33 and 36°C groups.
The number and labeling index of type A
spermatogonia were not affected, which
indicates that the proliferative activity
of type A spermatogonia is normal at high
temperature. The number of cysts of
labeled type B spermatogonia was extremely
decreased. The fish which had an injec-
tion of BrdU were kept at 27 and 33°C, and
examined the most advanced stage of
spermatogenesis of the labeled germ cells.
The results showed that labeled spermato-
zoa appeared 5-9 days after the injection
at 27°C and 3-5 days at 33°C.
These results indicated that the dif-
ferentiation of type B spermatogonia is
specifically inhibited at high tempera-
ture.
ON THE SPERMATOGENESIS OF THE FRESHWATER
EARTHWORM, BRANCHIURA SOWERBYI. I. THE
FINE STRUCTURE OF THE CENTRAL MASS.
M.Shirasawa and N.Makino. Dept. of Biol.,
Tokyo Med. Coll., Tokyo.
The spermatogenesis of the freshwater
earthworm, Branchiura sowerbyi was stud-
ied by light and electron microscopy. Male
reproductive cells are released from the
testes as spermatogonia, and maturation
takes place in coelomic pouches called se-
minal vesicles. Male cells in every stage
of maturation make a morula-like cluster,
connected together by nucleus-less proto-
plasm, called central mass. Central mass,
which is thought to serve as a supporting
or nutritive struture for the male cell,
contains many small mitochondria, free ri-
bosomes, and granular or agranular endo-
plasmic reticulum. Mitochondria of the
central mass, which originate in spermato-
gonia, propagate during early stages of
maturation and seem to be stored as sup-
plies of mitochonria for the formation of
the chondriosome body in spermatids. An-
other principal organella of the central
mass is membrane-less, pyronin-positive,
large dense bodies. They seem to originate
from chromatoid bodies of spermatogonia
and are thought to concern with the pro-
duction of free ribosomes of the central
mass. Golgi apparatus was observed in the
central mass, though secretion was not yet
obvious.
LECTIN BINDING TO TESTIS OF CRICKET GRYLLUS
BIMACULATUS.
A.Suzuki, K.Nishimura and M.Obika. Dept. of
Biol., Keio Univ., Yokohama.
The sugar chain expression in germ
cells of cricket Gryllus bimaculatus during
the spermatogenesis was investigated using
fluorescent lectins or peroxidase conjugated
lectins. When spermatozoa and spermiogenic
cells smeared on a slide glass were treated
with fluorescein isothiocyanate-labelled
concanavalin A or rhodamine-labelled wheat-
germ agglutinin, acrosomal area was labelled
intensely but other part also reacted
weakly, as described in Drosophila and two
species of Orthoptera. Soy bean agglutinin
(SBA) bound very specifically to
spermatogonia and spermatocyte among
peroxidase conjugated six lectins (Con A,
WGA, PNA, UEA-1, SBA and DBA) tested for
their affinity to paraffin section of
testis. In young spermatids beginning the
tail elongation, a round or cup-shaped
organelle was the only structure that bound
SBA. The SBA binding figure disappeared
during the process of maturation of
spermatid to sperm. The organelle
corresponded to the structure of dictyosome
and acroblast by electron-microscopical
analysis.
Developmental Biology 1197
SPERM SURFACE AUTOANTIGEN INVOLVED IN
FERTILIZATION IN THE NILE TILAPIA
(Oreochromis niloticus).
K. Mochida, S. Adachi, K. Yamauchi and H. Takahashi.
Dept. of Biol., Fac. of Fish., Hokkaido Univ., Hakodate.
In the testis of fishes, several specific proteins are expressed
on the sperm plasma membrane during spermiogenesis. These
proteins heve been known as autoantigens which are isolated
from fishes' own immune system by the blood-testis barrier,
and may have specific functions during and after
spermiogenesis. In order to investigate whether the sperm
surface autoantigens are involved in fertilization, fertility of
sperm coated with anti-sperm autoantibody was examined in the
Nile tilapia. Moreover, partial purification of sperm
autoantigens was attempted in the present study.
Anti-sperm autoantibodies were isolated from serum of male
Nile tilapia immunized with allogeneic sperm emulsified with
Freund's complete adjuvant. Freshly spawned eggs were
inseminated with sperm reacted with autoantibody in artificial
seminal plasma. The fertlity of sperm coated with autoantibody
was prominently lower than that of sperm coated with non-
specific antibody. The result suggests that at least one of the
autoantigens plays an important role in fertilization in the Nile
tilapia.
Membrane proteins of sperm were solubilized with non-ionic
detergent, octyithioglucoside. The extract was loaded onto the
affinity column using anti-sperm autoantiboy-Sepharose 4B, and
autoantigens were eluted with 8 M urea. The eluted fraction was
analyzed by SDS-polyacrylamide gel electrophoresis. Under
reducing condition, the fraction showed major polypeptide
bands centered at 80 kDa as well as some minor bands. By
immunoblotting analysis by autoantibodies, four main bands
containg the 80 kDa band were identified as autoantigens.
LOCALIZATION OF SPECIFIC RECEPTOR
FOR ARIS ON SPERMATOZOA OF STARFISH,
Asterias amurensis
A. Ushiyama', K. Chiba’ and M. Hoshi!
‘Dept. of Life Science and Gene Reserch Center, Tokyo
Institute of Technology, Yokohama.
Starfish egg jelly coat contains a highly sulfated
glycoprotein, which is called acrosome reaction-inducing
substance (ARIS). ARIS is essential for inducing the
acrosome reaction. We previously demonstrated that
FITC labeled ARIS bind specifically to homologous sperm,
indicating the presence of ARIS-receptors on the sperm.
By using two methods, we studied the distribution of
ARIS-receptors on the sperm surface. Competition of
FITC-ARIS binding was assayed with a head fraction
and a tail fraction of spermatozoa. Head fraction bound
FITC-ARIS specifically almost to the same extent as whole
sperm. Tail fraction did not bind FITC-ARIS appreciably.
ARI|S-binding was also assayed by microscopical
observation of intact spermatozoa after incubation with
fluorescent polystylene beads (Fluosphere) coupled with
ARIS. The binding of fluosphere coupled with ARIS was
specific and restricted to the anterior part of sperm heads.
Weconclude fromthese datathat ARIS-receptors localize
on the anterior surface of sperm heads.
THE ER PROTEIN, CALRETICULIN, IS PRESENT IN
THE ACROSOME OF POST-MEIOTIC CELLS IN RAT
TESTIS.
M.Nakamura! ,M.Moriya’ ,Y.Michikawa’ ,T.Baba‘’,
S.Okinaga’,T.Kobayashi’ and K.Arai'’. ‘Dept.
of OB/GYN,Sch.of Med.,Teikyo Univ.,Tokyo,
?Zool.Inst.,Fac.of Sci.,Hokkaido Univ.,
Sapporo, and *Inst.of Appl.Biochem.,Univ.of
Tsukuba, Tsukuba.
In this study, this protein has been
identified as a calreticulin. Immuno-stains
for the localization of this protein in rat
testis revealed that the protein is present
in the ER, the Golgi body and the acrosomal
vesicle of round spermatids, and in the
acrosome of sperm. The complete amino acid
sequence of calreticulin deduced from the
cDNA nucleotide sequence appeared to be
Similar to that of mouse calreticulin.
The cDNA encoded a protein of 416 amino
acids, but a 17-residue NH,-terminal signal
sequence was removed.The mature 399 residue
protein of Mr=46347 ends in Lys-Asp-Glu-Leu.
There were 7 amino acids replaced. When
Northern blot analysis, using RNAs prepared
from purified populations of rat spermato-
genic cells, was performed,the calreticulin
mRNA was found in pre- and post-meiotic
cells. Furthermore, the purified protein
has two Ca?* binding sites. These results
suggest that calreticulin, of which
biosynthesis is already started before
meiosis, plays a role in Ca’?* storage in
spermatogenic cells of rats, and that this
ER protein incorporates into the acrosomal
vesicle via the Golgi body without
glycosylation during spermiogenesis.
EFFECTS OF HORMONES ON THE TESTIS FRAGMENT
CULTURED IN VITRO IN THE MEDAKA.
M. Matsumoto and K. Onitake. Dept. of
Biol., Fac. of Sci., Yamagata University,
Yamagata.
We have reported that the spermatogenesis
progressed under the conditions of cell
culture of spermatocytes and organ culture
of testis fragments. In the present study,
effects of several hormones, human
chorionic gonadotropin:HCG, follicle stim-
ulating hormone: FSH, testosterone and 17-
Sestradiol, on the differentiation of
spermatogonia in the testis fragaments
cultured in L-15 medium supplemented with
fetal bovin serum. To identify the differ-
entiation process more cleary, BrdU was
added in the culture medium and BrdU in-
corporating spermatogonia were pursued
immunohistochemically using the anti-BrdU
monoclonal antibody. When FSH and 17-£8
estradiol were added, the number of sper-
magonial cysts increased and the differ-
entiation process from spermatogonia to
spermatocytes was observed. Addition of
HCG was also effective as same as FSH.
Testosterone was neither effective on in-
crease of the number of spermatogonial
cysts nor the differentiation of spermato-
gonia to spermatocytes. Furthermore, after
5days of culture, spermatids containing
BrdU appeared. These results suggests that
spermatogenesis in the testsis fragments
cultured in vitro was controlled by the
hormones and progressed as same as in vivo.
1198 Developmental Biology
IN VITRO SPERMATOGENESIS FROM THE PRIMARY
SPERMATOCYTES UNDER THE BROMODEOXYURIDINE
CONTAINING MEDIUM IN THE MEDAKA.
M. Suzuki and K. Onitake. Dept. of Biol.,
Fac.of Sci., Yamagata University, Yamagata.
We have revealed that the isolated pri-
Mary spermatocytes of the medaka completed
first and second meiotic divisions and
differentiated into functional sperm in
vitro. In the present study, in order to
get more direct evidence of the production
of functional sperm in vitro, primary
spermatocytes were precultured in L-15
medium supplemented with 10% fetal calf
serum containg BrdU of l1pM/ml for 3 hr.
After preculture, they were cultured in L-
15 medium without BrdU for 5 days. Process
of spermatogenesis was pursued immuno-—
histochemically using anti-BrdU monoclonal
antibody. After 2 days of culture, sperma-—
tocytes in l1yM BrdU/ml completed first and
second meiotic divisions and differenti-
ated into flagellated spermatids. Time
course of differentiation was almost same
as in the normal culture medium, while the
spermatocytes cultured in the medium
containing BrdU of 50y4M /ml required 4
days for completion of the first meiotic
divisions,but second meiotic divisions
never occured. These results indicate that
DNA syntheses at primary spermatocytes
were necessary for the completion of
second meiosis follwing further differ-—
entiation into spermatozoa.
DIFFERENTIATION OF SECONDARY
SPERMATOGONIA TO PRIMARY SPERMATOCYTES
IN ORGAN CULTURE OF TESTES FRAGMENTS
FROM CYNOPS PYRRHOGASTER: ANALYSIS OF
PROTEIN SYNTHESIS. ,
K. Maekawa and S. Abe. Dept. of Biol.
Sao MACS C1 SStlo A UMEMOLO Wrrsiys ,
Kumamoto.
We have previously shown that
secondary spermatogonia differentiate
into primary spermatocytes by FSH in
organ culture of testes fragments and
that FSH induces proliferation of
spermatogonia in testes fragments which
were incubated in a control medium for a
week(Ann. Meeting of Dev. Biol., 1992).
In this study, we examined if the
synthesis of new proteins is induced by
the addition of FSH in the testes
fragments which were cultuped for a week
in the absence of FSH. [~~S]methionine
was incorporated into the testes
fragments, the cell extracts were
electrophoresed in two dimensions, the
gels were exposed to an imaging plate
for 3-4 days. Analysis by an image
analyzer showed that at least two spots
(pI6.9, MW1.8K and pI5.5, MW1.3K) were
detected to appear within 1 day after
the addition of FSH. These results
indicate that FSH triggers the synthesis
of some proteins in Sertoli cells and/or
secondary spermatogonia, which leads to
the proliferation of spermatogonia.
DIFFERENTIATION OF PRIMARY SPERMATOCYTES
TO ELONGATING SPERMATIDS BY FSH IN ORGAN
CULTURE OF TESTES FRAGMENTS FROM CYNOPS.
Z. S. Ji and S. Abe. Dept. of Biol.Sci.,
Fac. of Sci., Kumamoto Univ., Kumamoto.
To study if the processes in
spermatogenesis beyond primary
spermatocytes (PC) depend on hormones
and Sertoli cells, testes fragments rich
in PC were cultured in a chemically
defined L-15 medium supplemented with
various hormones and vitamins.
In a control medium a few percent of
cysts contained round spermatids but no
elongating spermatids on the second
week, while both of the diameter and
viability of germ cells decreased to
about 40%. On the other hand, ina
medium supplemented with various
hormones and vitamins more than 10% of
the cysts differentiated to elongating
spermatids and both the diameter of
cysts and viability of cells were
maintained at the initial level.
Selective removal of hormones and
vitamins revealed that FSH alone
promoted the differentiation but neither
androgens nor vitamins did. This is
consistent with our previous results
that FSH binds to the newt testes in PC-
stage as well as in spermatogonia-stage.
This result indicates that FSH and
Sertoli cells play a major role in
initiating nuclear elongation of
spermatids.
STABILITY OF MANCHETTE (MICROTUBULE
BUNDLES) IN NEWT ELONGATE SPERMATIDS.IV.
Y. Mazaki, K. Takamune and S. Abe.
Dept. of Balol. Sci) RalceaOmepotneery
Kumamoto Univ., Kumamoto.
We have previously shown that
Manchette structure in newt elongate
spermatids(ET) is resistant to the
treatment of Ca“* and low temp and that
1M NaCl sup from ET-rich testes contains
MAPs which promote tubulin
polymerization and confer the low temp-
resistance to the microtubules(Ann.
Meet. Zool. Soc., 1991).
Copolymerization of porcine brain
tubulin with the 1M NaCl sup and porcine
brain MAPs(pbMAPs) showed that the
amount of 76K and 59K of 1M NaCl sup
bound was reciprocal to that of pbMAPs
bound, suggesting that the binding sites
On microtubules for 76K and 59K were
common for pbMAPs. Western blotting of
cell extracts of purified populations of
primary spermatocytes(PC), round
spermatids(RT), ET and mature sperm(MS)
with anti-76K and anti-59K as probes
showed that 76K was present through PC
to ET but was absent in MS and that no
59K was present through all stages.
These results indicate that 76K is
responsible for promoting polymerization
of tubulin and resisting disassembly and
that it is somehow regulated so that 76K
does not confer low-temp resistance to
microtubules in PC and RT in vivo.
Developmental Biology 1199
HOW IS THE FLAGELLAR LENGTH OF MATURE
SPERM DETERMINED? VI. QUANTITATIVE
ANALYSIS OF TUBULIN SYNTHESIS RATE IN
SYNCHRONOUS CULTURE OF ROUND SPERMATIDS
FROM CYNOPS AND XENOPUS. i
A. Yoroisaka, K. Takamune and S. Abe.
DAPEG O8 BWiOGl, SEilop WAGES O88 Sto,
Kumamoto Univ., Kumamoto.
We have shown that the difference in
the period of flagellar growth between
Cynops and Xenopus may be due to the
difference in the period of tubulin
synthesis between the two species(Ann.
Meet. Zool. Soc. 1990).
In this study, we measured tubulin
Synthesis rate quantitatively in
Synchronous culture of round spermatids
from both species: [ 5s )}methionine was
incorporated to round spermatids, the
proteins extracted were electrophoresed,
the gels were exposed to an imaging
plate and the intensity of the tubulin
band was analyzed by an image analyzer.
In Cynops, tubulin synthesis rate
rose up to about 150% by day 3 and fell
down to about 40% of the initial value
by day 6. Then, the value declined
slightly by day 12 but about 20%
remained on day 12. On the other hand,
in Xenopus, the synthesis rate on day 3
was almost the same as the initial one
but then it dropped to reach 5% on day
9. These results demonstrate and confirm
the previous results that tubulin
synthesis in Cynops round spermatids
continued longer than in Xenopus.
GENE ANALYSES OF THE SPERM-SPECIFIC BASIC
PROTEIN IN, XENOPUS LAEVIS.
N.Ariyoshi', K. akamune , S-Abé!, K.Mita2,
and Ch.Katagiri~. Dept.Biol. Cop Meo
Sci., Kumamoto Univ., Kumamoto, “Zool.
Inst., Fac.Sci., Hokkaido Univ., Sapporo.
The chromatin proteins of the mature
sperm of Xenopus laevis comprise six
sperm-specific nuclear basic proteins
(SP1-6) in association with four types of
core histones. Southern analyses of the
genomic DNA from liver of the Xenopus J
strain using SP4 cDNA as a probe revealed
that four fragments (12Kbp, 7Kbp, 4Kbp and
2Kbp) of the EcoRI digests and two frag-
ments (15Kbp and 10Kbp) of the HindIII
digests contain SP4 genes. Mapping of both
SP4 genes and restriction enzyme sites on
the 12Kbp fragment of EcoRI digest indi-
cated the existence of three loci for the
SP4 genes and a HindIII site on one of the
intermediate regions between the SP4
genes. These results suggested that two
hybridizable fragments with SP4 cDNA of
HindIII digest are linked together, so
that plural SP4 genes are arranged over
about 25Kbp area. S1 nuclease mapping and
complete base sequences of one of these
SP4 genes and its flanking region showed
that TATA box and CCAAT homology (opposite
strand) were 28bp and 84bp nucleotides
upstream from the transcription start
site, respectively. The SP4 gene had a
single, short intervening sequence in
contrast with the rooster and trout
protamine genes which are intronless.
ANALYSIS OF NUCLEAR BASIC PROTEINS AND
CHROMATIN STRUCTURE OF RANA CATESBEIANA
MATURE SPERM
Meee Olle pan Kae Ohsumi2, K. Takamune?, and Ch.
Katagiri. Zool. Inst., Hokkaido Univ.,
Lab. Cell Dev. Biol., Tokyo Inst.
Technol., “Dept. Biol., Kumamoto Univ.
Previous studies reported that the
testis proteins in Rana catesbeiana contain
testis-specific histone Hl and four core
histones (H2A, H2B, H3, H4). Analyses on
acetic acid/urea/Triton X-100 (AUT)-PAGE of
acid extracts from mature sperm separated
by Percoll density gradient centrifugation
showed sperm-specific proteins besides core
histones. Fractionation of these proteins
using reversed-phase HPLC and SDS-PAGE and
their amino acid analyses revealed three
sperm-specific, very lysine-rich Hls and
four somatic core histones. Incubation of
sperm in nucleoplasmin (NP) resulted in
pronounced nuclear decondensation and loss
of sperm-specific H1ls but not core
histones. Sperm chromatin was totally re-
sistant to micrococcal nuclease digestion,
but yielded 150- and 350 bp nuclease-
protected DNA fragments after incubation
with NP. Electron microscopy of NP-treated
sperm nuclei showed beaded fibers
possessing variable linker lengths. These
results suggest that Rana sperm chromatin
contains nucleosome-like structure with
repeat lengths different from that of
typical somatic chromatin.
cDNA CLONING AND EXPRESSION OF ACTIVIN B IN THE
TESTIS OF JAPANESE EEL, ANGUILLA JAPONICA
T. Miural, M. Tanaka, K. Yamauchi2, M. Asashima3 and Y.
Nagahama’.
‘Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki,
2Fac. of Fisheries, Hokkaido Univ., Hakodate and 3Dept. of
Biol., Yokohama City Univ. Yokohama
A cDNA insert encoding activin B was isolated from a
Japanese eel testis cDNA library. The nucleotide sequence of
eel activin B cDNA is 3.3 kb long. The insert contains an
open reading frame predicted to encode a protein of 395
amino acid residues, of which the C-terminal 115 residues
represent the activin B-subunit proper. The deduced protein
of the activin B subunit has 89% homology with that of the
human and chicken subunit, and 88% homology with that of
the pig and Xenopus subunit. Northern blot analysis of testis
RNAs using this cDNA insert demonstrated a single mRNA
species approximately 3.3 kb in length. Testis expression of
activin B mRNA was very low or undetectable prior to HCG
treatment, was highest one day after HCG treatment and
declined as spermatogenesis progressed. The tempora
pattern of expression of activin B mRNA after HCG injection
is strongly suggestive of a role for activin B in early
spermatogenesis in the eel.
1200 Developmental Biology
ANALYSES OF PROTEIN-DNA INTERACTIONS IN
THE PROMOTER AND INTRON REGIONS OF THE
MOUSE AND HUMAN HAPLOID-SPECIFIC Hsp70
HOMOLOGUE GENES
H.Ando, Y.Saijoh, Y.Itol, J.Higuma and
H.Fujimoto, Lab. of Cell Biol., Mitsubi-
shi Kasei Inst. of Life Sci., Machida-shi,
Tokyo (1univ. of Hiroshima, Hiroshima).
Recent advances of molecular biological
techniques revealed that some of genes are
exclusively expressed in the post-meiotic
cells. We cloned mouse and human haploid-
specific Hsp70 homologue genes, hsc70t and
hum70t, respectively. To investigate
mechanisms of haploid-specific gene
transcription, genomic structures of both
genes were analysed. They had only one in-
tron in the 5' untranslated region and
their genomic organizations were con-
served. After determination of a
transcriptional starting point of the
mouse hsc70t gene, nucleotide sequences of
both genes were aligned in the promoter
and intron regions. Several stretches of
sequences were shared between mouse and
human genes. The interaction of mouse
nuclear proteins to these stretches was
analysed by the gel shift assay. A DNA-
protein complex caused by one of these
stretches was shifted in nuclear proteins
extracted from adult mouse germ cells,
but not from somatic tissues examined nor
younger mouse testis without spermatids.
Functional significance of these regions
remains to be determined by analyses of
transgenic mice.
EFFECTS OF ASCORBIC ACID PHOSPHATE ON THE
AUTOCRINE GROWTH OF CHONDROGENIC CELLS OF
THE CHICK EMBRYO
T.Miyazaki, K.Watanabe, M.Amano
Cell and Developmental Biology Laboratory,
Faculty of Integrated Arts and Sciences,
Hiroshima University, Hiroshima.
Perichondrial cells, the scleral fibro-
blasts of chick embryo, proliferated in
protein-free culture. The cells died
within a week after reaching stationary
phase. Ascorbic acid phosphate(Asc-P;
mixture of 2- and 3-phosphates) rescued
this type of cell death.
Four growth promoting factors(A-I~ A-IV)
were detected by DEAE-Sepharose chromato-
graphy in the conditioned medium obtained
from Asc-P containing culture. These were
different from previously described SAF-I
and SAF-II separated from the conditioned
medium without Asc-P.
A-II and A-IV manifested growth-promoting
activities to the scleral fibroblasts and
the scleral chondrocytes, but A-I did only
to the scleral fibroblasts. A-III showed
the activity only to the scleral chondro-
cytes. They promoted growth, but did not
promote cartilage differentiation.
These results suggested Asc-P altered
secretion patterns of growth factors. The
perichondrial cells were considered to
produce multiple autocrine growth factors,
which played roles on the own cells and ma-
tured chondrocytes differently in autocrine
and/or paracrine manner.
VIMENTIN EXPRESSION DURING THE DEVELOPMENT
OF SOMATOPLEURAL MESODERM OF THE CHICK
EMBRYO.
K.Hayashi, Y.Hagiwara and E.Ozawa. Div. of
Cell Biology, Natl. Inst. of Neuroscience,
NCNP, Tokyo.
The limb fields are eStablished by an
unknown inhibitory change on the limb-
forming potential of the flank mesoderm.
We obtained a monoclonal antibody that
reacts to the flank mesoderm of the stage
22 chick embryo, but not to the mesoderm
of neck, wing bud, leg bud and tail.
Further investigation of this antibody
revealed that the antigen iS vimentin.
Vimentin expression during the development
of the somatopleural mesoderm was studied.
All the mesodermal cells expressed
vimentin at stage 10. But the expression
disappeared at the somatopleural mesoderm
by stage 16. The flank somatopleural
mesoderm re-expressed vimentin at stage 18
at the dorso-medial region, and the whole
flank mesoderm expressed vimentin at stage
20. On the other hand, the limb bud
mesenchymal cells did not re-express
vimentin until stage 27 (about 2 days
later). Considerably earlier onset of
vimentin re-expression at the flank region
May be a useful molecular marker for the
study on the establishment of the limb
fields.
THE EXPRESSION PATTERN OF Chox—] GENES
IN THE TRUNK OF CHICKEN EMBRYOS.
Y.Yokouchi_ and A.Kuroiwa, Dept. of Cell
BiCIoe ys Res. Inst. for Tuberculosis &
Cancer. Tohoku Univ. Sendai.
We have already reported that the expression pattern
of homeobox genes belonging to Chox—I cluster are
closely related to the segmental structure of the carti-
lage along proximo-—distal axis in developing chick
limb bud. ;
We investigated the expression patterns of these
genes in the trunk of embryos. In somite derived tis—
sues, Chox—].8 was expressed between somite 26 and
tail. On the other hand, Chox- J.9 was expressed
between somite 29 and tail end. In contrust, Chox-
1.10 was not expressed in somite derived tissues.
In neural tube, Chox—1.8 and Chox—1.9 were ex—
pressed at the same position as somatic expression
were found, and Chox—J.10 was expressed from the
region correspondent to somite 33 to tail end.
In the gut, we found that these genes were expressed
along the anteroposterior axis correlated to the func—
tional domains of the gut. Chox—1.8 was expressed in
the mesoderm of the cecum, Chox-J.9 was expressed
in the mesoderm of large intestine and Chox-J.10 was
expressed in the mesoderm of the cloaca and the
endoderm of the hind gut.
These results suggested that these pene might be
responsible for the determination of the positional
specificities along the anteroposterior body axis.
Developmental Biology 1201
BASIC FGF MAINTAINS THE CHARACTER OF THE
PROGRESS ZONE CELLS OF ST.20 CHICK LIMB
BUDS IN CULTURE.
A.Watanabe,K.Ohsugi and H.Ide. Biol-.Inst.,
Fac.of Sci.,Univ. of Tohoku, Sendai.
Distal region (progress zone,PZ) of
chick limb bud is’ important for the
cartilage pattern formation, in which
mesodermal cells are Maintained in an
undifferentiated and rapidly growing state
by the interaction with the apical
ectodermal ridge (AER). It is known that
the AV-1 antigen expresses in distal-
anterior region and Chox.7 gene expresses
in distsal region of st.20 chick limb bud
and these expressions depend on the AER.
We have already reported that bFGF
activated AV-1 expression in cell culture
of st.20 chick limb buds by the combina-
tion with the BRL3A conditioned medium
(BRL3A-CM).Growth of the PZ cells is
stimulated and cartilage differentiation
is inhibited under this condition.
In this study, we found that bFGF
maintained Chox.7 expression in cell
culture of st.22 limb buds, and affected
growth and cartilage differentiation in
the cell culture of st.20 and st.22 limb
buds by the combination with the BRL3A-CM.
Furthermore,it was indicated that after
bFGF treatment, the grafted PZcells
fitted well with limb cartilage pattern
in vivo. These indicate that bFGF is
effective for the maintenance of the PZ
character at molecular and cellular level
in vitro, and that bFGF may act as a PZ
Maintenance factor in vivo.
MRC-5 CELLS CAUSE A-P DUPLICATION IN
CHICK LIMB BUD.
S.Yonei-, K.Tamura!, E.Koyama“, S.Noji%,
H.Ide~.
Biol. Inst., Tohoku Univ., Sendai!, Dept.
Biochem Okayama Univ. Dental School,
Okayama and Dept. Biol. Scj. and Tech
nol. Tokushima Univ. Tokushima~.
Grafting of the polarizing region (ZPA)
to the anterior margin of chick limb bud
leads to mirror-image duplication of
Cartilage pattern. In this study, we found
that MRC-5 cells (normal human lung fibro-
blast) showed the polarizing activity when
grafted to the anterior region of chick
leg field at pre-limb bud stages (st.14 -
st.17). MRC-5 cells could induce addition-
al digits and some duplications at
tibia/fibula level. However, the activity
attenuated in stage 18 leg buds and disap-
peared in stages 19-20 leg buds. Chimera
analysis showed that slow growth of MRC-5
cells occurred in the leg bud and the
cells located proximal region. When the
cells were implanted into the anterior
region of wing field, the percentage of
duplicate formation was very low.
These results suggest that products of
MRC-5 cells are involved in three possible
processes of the duplicate formation
that is, induction of polarizing activity,
maintenance of the activity which is
present weakly at pre-limb bud stages, and
determination of antero-posterior axis as
the ZPA factor(s).
DIFFERENTIATED LIMB CARTILAGE CELLS
CAN PARTICIPATE IN LIMB CARTILAGE
PATTERN FORMATION ACCORDING TO THEIR
POSITIONAL VALUES.
M. Sato and H. Ide
Biol. Inst., Tohoku Univ., Sendai.
In developing limb bud, the cells
emerging from progress zone participate in
limb pattern formation according to their
allocated positional values.
To examine whether differentiated
cartilage cells retain their positional
values, we grafted tissue blocks of chick
leg cartilage, femur and metatarsus, to
the progress zone of stage 20 quail wing
buds. Both cartilage tissues participated
in the host wing cartilage pattern
formation, and the grafts of metatarsus
localized in more distal region than those
of femur, suggesting that the limb
cartilage cells may keep their original
positional values along PD axis.
When non-limb cartilage tissue, scapula,
sternum, rib or sclera, was grafted to the
wing buds, scapula and sclera participated
in wing pattern formation but rib and
sternum, which were of somite origin, did
INOVIE c Mixed cultures with these
differentiated chondrocytes and limb bud
cells revealed that sternum cells and limb
bud cells segregated one another although
sclera and femur cells were intermingled
with the limb bud cells, suggesting that
the difference in cell adhesiveness may be
a cause of difference in the participation
in limb cartilage pattern formation.
THE PROXIMALIZATION BY RETINOIC ACID IN
DEVELOPING CHICK LIMB BUD. 2
K.Tamyra™, Y.Yokouchi“~, A.Kuroiwa
H.Ide-.
Biol. Inst.1 and Res. Inst. for TB and
Cancer Tohoku Univ., Sendai.
In regenerating urodele limbs, retinoic
acid (RA) has been known to proximalize the
positional value, resulting in the forma-
tion of tandemly repeated limbs along the
proximo-distal axis. However, there is no
evidence which supports the proximalization
by RA developing limb bud at present.
When the beads pre-soaked in RA are
implanted into the anterior margin of chick
limb buds, they posteriorize limb bud
tissues and induce ZPA. In order to estab-
lish the proximalization by RA in the chick
limb buds, 24 hr after implantation of the
RA beads to stage 20 wing bud, the tissue
blocks which contain new ZPA induced by RA
and the linked distal cells were trans-
planted into other wing buds at stages 16-
17. Following this operation, the tissues
often developed a complete set of limb
cartilage elements along proximo-distal
axis from humerus to digits (control grafts
adjacent to the DMSO-containing beads
formed no cartilage structure). These
ectopic structures were found to consist of
graft tissue by chimera analysis, indicat-
ing that the limb bud tissues under the
influence of RA could form more proximal
elements than the presumptive ones by the
transplantation to early wing buds. The
proximalization was also supported by the
expression pattern of Chox-1 genes.
and
1202 Developmental Biology
FETAL WOUND HEALING. EXPRESSION OF SOME
DIFFERENTIATION MARKERS AT THE WOUND SITE.
S. Ihara and Y. Motobayashi-. Dey. Biol.
Lab., Dept. of Plast. Surg., and ~Dept. of
Biochem., Kitasato Univ. School of
Medicine, Sagamihara.
We have studied by in situ hybridiza-
tion the expression of several differen-
tiation markers at the wound site in day-
17 fetal rat skin 24 h after intrauterine
wounding. Oligonucleotide probes (40-mers)
were synthesized for the following mark-
ers: Bactin; 60k and 67k type II kera-
tins; a1l(1I) and al(III) procollagen; and
C-terminus and EIIIA segments of fibro-
nectin. Feasibility of the probes was con-
firmed by Northern analysis with poly(A)-
RNA prepared from day 16-20 fetal and
newborn rat skin, although the expression
of 60k keratin was exceptionally undetect-
able at all stages. The expression of £8 -
actin was ubiquitous and its signal inten-
sity was unchanged during normal skin mor-
phogenesis, but strong signals were seen
at the upper half epidermal layer of the
wound edge. Unlike widespread expression
of 60k keratin, the expression of 60k
keratin was restricted to the lower half
epidermal layer close to the wound edge.
No special induction of the two types of
collagen was observed, whereas panniculus
carnosus and epidermis expressed EIIIA*
fibronectin predominantly at the wound
edge. The present results suggest a delay
of differentiation at the wound sites.
PRODUCTION OF MONOCLONAL ANTIBODIES
AGAINST MOUSE EMBRYONIC SUBMANDIBU-
LAR GLANDS.
Y. Mori’, Y. Hieda!, Y. Kano”, T. Morita! and Y. Nakanishi’.
1Dept. Biol., Coll. Gen. Educ., Coll. Bio-Medical Tech.,
Osaka Univ., *Dept. Chem., Fac. Sci., Nagoya Uniy.
The morphogenesis of the mouse embryonic submandibular
gland, lung and kidney has been proved to proceed under the
influence of epithelial-mesenchymal interactions. We have
tried to produce monoclonal antibodies against mouse or-
gan rudiments to find novel extracellular matrix components,
which would be involved in such interactions, and also to ob-
tain valuable epithelial markers for the investigation of the
morphogenetic movement. Embryonic submandibular glands
or the mixture of submandibular glands, lung and kidneys
were injected intraperitoneally into rats, and the splenocytes
were fused with mouse myeloma cells. Hybridoma media
were assayed by staining cryostat sections containing sub-
mandibular gland, lung and kidney. More than 60 mono-
clonal antibodies against components of basal lamina, mes-
enchyme and epithelium were obtained. No obvious modula-
tion with these antibodies of the epithelial morphogenesis of
submandibular gland has been observed yet. Among those,
mAb 65 showed a unique, fibrous staining at the basal lam-
ina areas and the basal part of clefts. Concentrated mAb 65
stained clearly the extracellular matrix of the mésenchyme,
suggesting that the antigen would be of mesenchymal nature.
Interestingly, mAb 65 stained roofplate area of the neural
tube in trunk region of the 13-day mouse embryo in addition
to the extracellular matrix of bovine and mouse skin. Im-
munoblot analysis showed that mAb 65 recognized a band of
MW more than 400 kD.
GLYCOSAMINOGLYCANS AND MORPHOGEN-
ESIS OF MOUSE EMBRYONIC SUBMANDIBU-
LAR EPITHELIUM.
Y. Nakanishi!,Y. Mori?, K. Yoshida?, T. Morita?, and Y.
Fukuda*. 'Coll. Bio-Medical Tech., 7Dept. Bicl., Coll. Gen.
Educ., Osaka Univ., *Seikagaku Kogyo Ltd. and *Dept. Pa-
thol., Nippon Medical Sch.
The initial branching morphogenesis of the mouse embryonic
submandibular epithelium has been suggested to be regulated
by extracellular matrix components including glycosamino-
glycans. Immunohistochemical studies with monoclonal an-
tibodies against intact and chondroitinase-degraded chon-
droitin sulfate chains showed that they were located at the
extracellular spaces of the mesenchyme and epithelial cell
mass in addition to basal lamina. Basement membrane type
heparan sulfate proteoglycan (HSPG) was found in the basal
lamina. Chondroitinase ABC in the medium did not inhibit
the initial branching of the late 12-day gland though chon-
droitin sulfate chains in the tissues were almost removed by
the enzyme. In contrast, heparitinase I brought about a sig-
nificant inhibition of the DNA synthesis and complete in-
hibition of the initial branching of the late 12-day gland.
Matrigel-clotted epithelium cultured under the influence of
the mesenchyme did not undergo a specific branching mor-
phogenesis in the presence of heparitinase I. Collagen IV and
HSPG core protein in the heparitinase-treated glands were
found both in the epithelial cell mass and in the basal lam-
ina. Transmission electron microscopic study indicated that
intimate contacts between the mesenchymal and epithelial
cells were much fewer in the heparitinase-treated glands than
in the controls. These results suggested that changes in the
basal lamina architecture altered the growth and morphology
of the epithelium and epithelial-mesenchymal interactions.
ELASTIN GENE EXPRESSION PATERN ON FIBROBLASTS
OF FASCIA DURING MOUSE ORAL DEVELOPMENT.
T.Yamaai, Dept. Oral Anat., Okayama Univ. Dent. Sch.,
Okayama.
Fascia is a term so vague in usage that it
signifies envelopes of muscles, nervefibers, vessels,
renal and splenic tissues. The arrangement of such
fascia is highly valuable for sustainment and
isolation of tissues. Elastin is the major component
of elastic fibers and is found abundantly in fascia.
Prior to the post fixation by a microwave
processor for 5min at 37°C , C3H mice were fixed by
perfusion with 4% paraformaldehyde. The gene
expression of elastin in fascia of perinatal mice was
investigated by a simplified in situ. hybridization
method with 35S labeled riboprobes on 5um thick
serial sections. After 10-day exposure, signals were
observed on autoradiograms.
Signals of the anti-sense probe were observed
on fibroblasts of fascia from 16-day fetus to 2-week
neonate. The signal intensity in fascia of buccal
glands increased synchronously and became
maximum at 1-week neonate. However the signal
intensity in dermis, perimysia, periostea and
perineuria increased asynchronously. The signal
intensity of signal detectable fibroblasts in these
tissues was not homogeneous. The intense, weak or
no signal detected cells could be observed at same
stage. The signal was more intense on the portion
which had higher mobility in these tissues. It was
suggested that the elastin gene activation was
influenced by degree of intensity of occlusal
stimulation and stimulation from outside.
Developmental Biology 1203
MORPHOGENESIS OF THE TONGUE IN THE FROG
LARVA ( Rana tagoi )-
Masahiko Kumakura and Kan Kobayashi. Dept.
Anat., School of Dentistry at Niigata,
Nippon Dent. Univ., Niigata
The process of lingual development in
R. tagoi was observed by using the light
and electron microscopies. At the stage of
first appearance of hindlimb bud in larva,
a pair of finger-like protrusion was
appeared in the bottom of the oral cavity
before the beginning of lingual
development. Lingual development began at
the time when the hindlimb bud was well
developed. At this stage, epithelial cells
surrounding the finger-like protrusions
prolifelated, and a butterfly-like
primitive tongue was appeared. A typical
shape of tongue of the frog was formed at
the stage of emergence of the forelimbs.
In this time, numerous cone-like papillae
were distributed densely on the lingual
dorsal surface. During shortening of the
tail, the fungiform papillae were begun to
form. In the process of fungiform
papillary differentiation, microvilli
first appeared on the cell surface of the
top of the cone-like papilla.
Subsequently, they differentiated into
microridges and, as a result, sensory disk
was completed. At the final stage of
metamorphosis, fungiform papillae showed
fundamentally similar structure to that of
adult form, while structures of filiform
papilla and lingual gland _ were still
remained as immatured compared to those of
adult frog.
ULTRACYTOCHEMICAL STUDIES OF PROTEOGLYCANS IN THE
ANURAN LARVAL NOTOCHORD.
F.Sasaki, J.Hirata, S.Nishikawa, *H.Iseki. Dept.
of Biol., School of Dent. Med., Tsurumi Univ.,
Yokohama. *Tokyo Med. and Dent. Univ., Tokyo.
During metamorphosis an anuran larval noto-
chord degenerates both in body and tail. The
immunohistochemical and ultrastructural localiza-
tion of proteoglycans were studied in the noto-
chord of Rana japonica and Bombina orientalis.
The notochordal sheath and the peripheral region
containing connective tissue were strongly la-
beled with anti-chondroitin-4-sulfate. At the
ultrastructural level, we examined the presence
of sulfated proteoglycans in the extracellular
matrix of notochord using the cationic dye poly-
ethyleneimine (PEI) and cationic colloidal gold
(CCG). After staining with PEI, the proteogly-
Cans appeared as granules in the extracellular
matrix of fibroblast, chondroblast, and chondro-
cyte. PEI-positive material was removed after
treatment of the tissue with chondroitinase ABC.
The labeling with CCG at pH 1.0 was also observed
on the chondroblast matrix and notochordal
sheath. The present observations suggest that
PEI-positive structures and CCG labelings repre-
sent sulfated proteoglycans aggregates.
OBSTRUCTION EFFECTS OF RETINOIC ACID ON
METAMORPHIC RECONSTRUCTION OF LOWER JAW OF
BROWN FROG, RANA ORNATIVENTRIS.
S. Kurabuchi. Dept. of Histol., Sch. of
Dentistry, Nippon Dental Univ., Tokyo.
Rana ornativentris tadpoles were used in
the current study. Retinoic acid (RA), 50,
5 or 0.5 uwg/weight g, dissolved in dimethyl
sulfoxide, was injected into the individ-
uals one time at the metamorphic crimax
stages. When the metamorphosis completed,
the gross-structural abnormality of the
body was searched. Thus, the RA treatment
resulted in the delay of metamophosis and
the reduction defects of lower jow. Such
malformations were frequently yielded in
the group of the RA 50 ug/g injected at the
stage XX (Taylor and Kallros, '46). The
victria blue-stained whole-mounts for visu-
alization of the morphology of cartilages
(Bryant and Iten, '74) showed that the
hyoid and the mandibla were undersized or
partially lost in such malformed lower
jaws. Furthermore, when the labeling of
5-bromodeoxyuridine (BrdU) was detected by
immunohistochemistry(PAP method), the BrdU-
immunoreactive cells remarkably decreased
about 6-12 hours after RA-injection
especially in the cartilages of the lower
jaw. These results suggest that the RA may
obstruct the reconstruction of the cartila-
genous skeletons from the larval form to
the adult form, therefore may cause
abnormal morphogenesis.
ANAYSIS OF METAMORPHIC CHANGES IN SMALL
INTESTINE OF XENOPUS LAEVIS BY
MONOCLONAL ANTIBODY
T. Sano, A. Kawahara, and M. Amano
Cell and Developmental Biology, Faculty
of Integrated Arts and Sciences,
Hiroshima University, Hiroshima
Amphibian metamorphosis is regulated
by thyroid hormones and prolactin (PRL).
Amphibian small intestinal epithelium
changes from larval type to adult one at
metamorphic climax stage. To define
the role of these hormones, we raised a
monoclonal antibody specific to adult
epithelium (A5D9 antibody).
Fragmented small intestines of larva
at st. 56-57 were cultured and treated
with T3 and/or PRL. The results showed
that A5D9 antibody recognized only an-
terior intestinal epithelium which had
been treated with T3 for five days. On
the other hand, PRL inhibited this T3-
dependent production of A5D9 antigen.
Next, we examined whether A5D9 antigen
could be also induced in vivo by T3
treatment for five days. A5D9 antigen
was not able to be detected in this as-
say, indicating that T3 activity was
suppressed in vivo.
The results suggested that the
metamorphic transition of intestinal
epithelial cells was directly promoted
by thyroid hormones, but suppressed by
PRL activity.
1204 Developmental Biology
DIFFERENTIATION OF CONNECTIVE TISSUE CELLS
OF THE XENOPUS SMALL INTESTINE CULTURED IN
THE ABSENCE OF EPITHELIUM.
A.Ishizuya-Oka and A.Shimozawa. Dept .of
Anat.,Dokkyo Univ. Sch. of Med., Tochigi.
We have previously reported that cells
originating from the larval connective
tissue (CT) are involved in metamorphic
epithelial changes in the anuran small
intestine; an aggregation of fibroblasts
rich in rough endoplasmic reticulum (Fr)
and macrophages (M@) participate in the
adult epithelial development and the lar-
val epithelial degeneration, respectively.
In the present study, we investigated
influences of the epithelium on differen—
tiation of Fr and M@. The epithelium was
removed by dispase treatment from tissue
fragments isolated from the anterior part
of the Xenopus small intestine at NF stage
57. Some of them were recombined with the
epithelium (CT+E), and the others remained
epithelium-free (CT-E). After cultivation
in the presence of thyroid hormone, the
number of Mgd@ that showed non-specific
esterase activity was significantly larger
in CT+E than in CT-E. The aggregation of
Fr was observed just beneath the epithe-
lium in CT+E, but was not observed in CT-E
at alle These results indicate that the
epithelium influences differentiation of
CT cells, which in turn influences epithe-
lial changes in the small intestine during
anuran metamorphosis.
cDNA CLONING OF COLLAGEN OF BULLFROG TADPOLE
(Rana catesbeiana)
S. Yomori, K. Oofusa and K. Yoshizato
Molecular Cell Science Lab., Zool. Inst.,
Fac. of Sci., Hiroshima Univ., Higashi-
hiroshima
Collagens are major proteins of
extracellular matrices and play critical
roles in the process of formation of
tissues and organs. The metabolism of
collagen is markedly activated in the
process of metamorphosis. In this study,
the synthesis of collagen during
metamorphosis of bullfrog tadpoles was
examined by RNA blot analysis and cDNA
cloning was performed. Using the human
al(I) collagen cDNA and al(II) collagen
gene as a probe, RNA blot analysis was
performed for measurement of collagen mRNA
levels in the tail of tadpoles. Both typel
and typeII collagen mRNA levels were
reduced by thyroid hormone. In contrast,
they were up-regulated by prolactin that
delays metamorphosis of frog tadpoles.
A cDNA library was constructed from
poly(A)* RNA obtained from tail of
bullfrog tadpoles. Fifty four positive
clones were obtained by plaque
hybridization using the human al(1)
collagen cDNA as a probe. The insert size
of one of these clones named G6A-1 was
largest and this clone was subjected to
DNA sequencing.
CLONING AND CHARACTERIZATION OF cDNA AND
GENE FOR THE BULLFROG TADPOLE COLLAGENASE
K. OOFUSA, S. YOMORI and K. YOSHIZATO
Molecular Cell Science Laboratory,
Zoological Institute, Faculty of Science,
Hiroshima University, 1-3-1 Kagamiyama
Higashi-hiroshima 724
A cDNA clone encoding the entire
sequence of bullfrog (Rana catesbeiana)
tadpole collagenase was fsolated from
cDNA library constructed from the
bullfrog tadpole back skin. This clone
(Tscl) was similar to both porcine and
human fibroblast collagenases; the
similarities of deduced amino acid
sequence were 78% and 65 %, respectively.
RNA blot analyses revealed that the
transcripts for collagenase increased in
both back skin and tall tissues of
thyrold hormone-treated tadpoles. In
addition, mRNA for collagenase was
decreased in tadpole talls by treatment of
prolactin.
Genome DNA was isolated from liver
and whole blood of adult bullfrog (female)
and were subjected to DNA blot analysis
using pTSCl as_ probe. A genome DNA
library was constructed from partially
digested DNA and plaque hybridization was
performed with pTSCl. Two positive
clones were Isolated from this. library.
Both of them carried about 20kbp Inserts
and subjected to restriction enzyme
mappings.
CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN DEPHOS-
PHORYLATION IN SPERMATOZOA OF SEA URCHIN
HEMICENTROTUS PULCHERRIMUS
T.Harumi, K.Hoshino and N.Suzuki. Noto
Marine Lab. Kanazawa Univ. Ishikawa.
When CHAPS-solubilized sperm proteins
were incubated with [7 -*?PJATP, a 48 kDa
protein was phosphorylated within 1 min.
Addition of cAMP or cGMP induced a rapid
dephosphorylation of the **P-phosphorylated
48 kDa protein. Half-maximal effective con-
centrations of cAMP and cGMP on the dephos-
phorylation were 0.3 » M and 4 yw M, respec-
tively. Protein phosphatase inhibitor,
calyculin A or okadaic acid inhibited the
dephosphorylation. TSKgel G3000SW chromato-
graphy of the CHAPS-solubilized sperm
proteins containing the *°*P-phosphorylated
48 kDa protein showed that the 48 kDa
protein was eluted in the fraction contain-
ing proteins whose molecular masses were
from 250 kDa to 400 kDa. Cyclic nucleotide-
dependent dephosphorylation activity for
the *?P-phosphorylated 48 kDa protein was
eluted in the fraction containing proteins
with molecular masses of over 400 kDa.
Photoactivatable cAMP analogue 8-Ns-
[°?7P]cAMP was incorporated into the 48 kDa
protein. These results suggest that H.
pulcherrimus spermatozoa possess cyclic
nucleotide-dependent dephosphorylating
activity for a *?P-phosphorylated 48 kDa
protein and the dephosphorylation occurs
upon binding of cyclic nucleotides to the
protein which probably exists as a hetero-
oligomer.
Developmental Biology 1205
CHARACTERIZATION OF TWO DIFFERENT PROTEIN
PHOSPHATASES IN SPERM TAILS OF THE SEA UR-
CHIN HEMICENTROTUS PULCHERRIMUS.
K.Hoshino, T.Harumi and N.Suzuki. Noto
Marine Lab. Kanazawa Univ. Ishikawa
It is known that receptor for sperm-
activating peptide-I (SAP-I) and guanylate
cyclase, which is a phosphoprotein, local-
ize in the sperm tails of the sea urchin
Hemicentrotus pulcherrimus. Upon treatment
of the spermatozoa with SAP-I, guanylate
cyclase looses its phosphate groups. In the
present study, using serial chromatography
on DEAE-Sephacel, Sepharose 6B and histone-
agarose columns we purified and charac-
terized two different protein phosphatases
(Type A and B ). Type A enzyme was isolated
from the particulate fraction of the sperm
tails with a solution containing 1% CHAPS.
The molecular weight of the enzyme was es-
timated to be 43,000 by gel filteration. An
apparent Km for dephosphorylation of 32P-
phosphorylated histone (%?P-histone) was
23.4 uw M and an apparent Vmax was 1.8 nmol
Pi/min. Okadaic acid inhibited the enzyme
activity with IDso of about 5x10-8M. Type B
enzyme was isolated from the soluble frac-
tion of the sperm tails. The molecular
weight of the enzyme was estimated to be
over 200,000 by gel filteration. An apppa-
rent Km for dephosphorylation of 32P-hist-
one was 23.0 uM and an apparent Vmax was
2.2 nmol Pi/min. The activity of type B en-
zyme was more sensitive to okadaic
acid.(IDso0; 1x10-°M).
CLONING OF CDNAS ENCODING GUANYLATE CYCLASE
AND SPERM ACTIVATING PEPTIDE-I RECEPTOR
EXPRESSED IN HEMICENTROTUS PULCHERRIMUS
SPERM
T.Shimizu, Y.Sendai, N.Suzuki. Noto Marine
Laboratory, Kanazawa University, Ishikawa
The H. pulcherrimus testis cDNA library
was constructed in Lambda gtl0, using
poly(A)+RNA isolated from testes collected
in November and December H. pulcherrimus
and oligo(dT) primer. As a result the cDNA
library containing 4.9x105 reconbinants was
produced. For screening cDNAs encoding
guanylate cyclase, oligo(dT)-primed cDNA
library from H. pulcherrimus testis in Lam-
bad gt10 were screened with two custom syn-
thesized oligonucleotides (45mer; AACAT-
GATCGCCATCATGGAGCGCTACACCAACAATCTGGAGGAG,
45mer ; GAGATTCTGCACGAGGAGTACTACGCGGGATTCGATC
CATGGGAC). Six clones were isolated with
two oligonucleotides. The longest clone had
an insert of 4171bp containing a poly(A)+
tail and open reading frame encoding 1125
amino acids. For screening cDNAs encoding
sperm activating peptide-I(SAP-I) receptor,
oligo(dT)-primed cDNA library from H. pul-
cherrimus testis in Lambda gt10 were
screened with custom synthesized oligo-
nuclotide (45mer;GGCATAGTCGTACGCACAGGG
ATGTGGGTATTGACCCTGCAACCA). Forty clones
were isolated with an oligonuclotide. The
major clone had an insert of 2443bp con-
taining a poly(A)+ tail and open reading
frame encoding 532 amino acids.
CHARACTERIZATION OF WHEAT GERM AGGLUTININ-
BINDING PROTEIN IN SPERM TAILS OF THE SEA
URCHIN HEMICENTROTUS PULCHERRIMUS
Y.Sato, T.Shimizu, Y.Sendai and N.Suzuki,
Noto Marine Lab. Kanazawa Univ. Ishikawa
A 220 kDa wheat germ agglutinin (WGA) -
binding protein was isolated from
0.1%Lubrol-solubilized fraction obtained
from sperm tails of the sea urchin
Hemicentrotus pulcherrimus by chromatog-
raphy on a WGA-Sepharose column and
preparative SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). The protein was
digested with protease V8. The resulting
protein fragments were purified by SDS-PAGE
and the partial sequence of a fragment
(VD2, 29kDa) was determined to be -Val-Ser-
Ser-Ile-Asp-Asn-Ile-Phe-Arg-Val-. The
sequence which was designated by the under-
line was identical to the conserved
sequence of H*-ATPase beta chain from
various sources including plants and
animals. A mixed antisense oligonucleotide
probe [5*’ GACACGGAAGATGTTGTCGATGCT (or
AGA)GCT(or GGA)GAC 3’] which corresponds to
the partial sequence was synthesized and
used for screening of 6x104 clones of a
cDNA library from H. pulcherrimus testes in
2 gt10. A cDNA (2.3kb) was isolated and the
nucleotide sequence was determined to have
2264 bases. An open reading frame predicts
a protein of 523 amino acids which is 85%
identical to rat H*-ATPase beta chain.
CLONING OF cDNAs ENCODING THE PUTATIVE
PRECURSORS FOR SPERM-ACTIVATING PEPTIDE
(SAPs).
H.Kinoh!, H.Fujimoto? and N.Suzuki!. ‘!Noto
Marine Lab. Kanazawa Univ. Ishikawa, ?Dept.
Orr Hol, Celi, Oi ee ke Sn, Winsiyo Ese
Tokyo, Tokyo.
The egg jelly is composed mainly of two
large acidic glycoproteins, a sialoglyco-
protein and a fucose sulfate glycoconjugate
(FSG). In the previous study, we
demonstrated that the polycolnal antibody
against FSG reacted with the accessary
cells and surface region of the oocytes in
the ovarian lumen.
In the present study, to investigate the
origin of SAP-I and its derivatives by in
situ hybridization, we cloned a cDNA en-
coding a putative precursor for SAP-I and
its derivatives in the sea urchin
Hemicentrotus pulcherrimus . About 3x104
clones of cDNA library from H. pulcher-
rimus ovary in A gtll was screened with a
32P random primed cDNA encoding for a
sperm-activating peptide precursor of
Strongylocentrotus purpuratus. Fourteen
clones were isolated, and the largest clone
had a 2.0kb insert, the remaining 13 clones
had a 1.3kb insert. Northern blotting
analysis demonstrated that the mRNA for the
1.3kb precursor was detected during whole
breeding season (from Nov. to Apri.).
However, the mRNA for the 2.0kb precursor
was detected only on January.
1206 Developmental Biology
PARTIAL PURIFICATION OF SPERM-ACTIVATING AND
SPERM-ATTRACTING SUBSTANCE IN THE ASCIDIAN,
CIONA INTESTINALIS.
M.Yoshida, K.Inaba, and M.Morisawa.
Misaki Marine Biological Station, Fac. of
Sci., Univ. of Tokyo, Kanagawa.
Spermatozoa of the ascidian, Ciona intes-
tinalis, exhibit chemotactic behavior to
eggs prior to fertilization (Miller,1975;
Yoshida and Morisawa, 1990). Here we tried
to purify sperm-activating and sperm-
attracting substance from unfertilized eggs.
The eggs were suspended and incubated in
artificial seawater for about 12 hr, and
supernatant obtained by brief centrifuge
(egg seawater) which exhibited strong sperm-
activating and sperm-attracting activies was
used as a starting material. The egg
seawater was lyophilized, then extracted by
absolute ethanol. After evaporation of
ethanol, the extract was applied successively
to reversed-phase (Sep-Pak C18), gel filtra-
tion (Bio Gel P-4), and cation exchange
(CM-Sephadex C-25) column chromatographies.
During these purification process, specific
activity of sperm activation and attraction
increased and both activities are always
co-migrated. These suggest that sperm-—
activating and sperm-attracting substance
is partially purified and both activities
are derived from the same molecule. Nature
of the substance is acidic, and molecular
weight of this molecule was estimated by
gel filtration as 2 ~ 2.2 kDa.
PURIFICATION OF SPERM-ACTIVATING PROTEINS
FROM UNFERTILIZED HERRING EGG. II.
S. Odat, H. Ohtake, yY. Igarashi®, K.
Sakai‘, Y. Shimizu? and M. Morisawa?.
imMisaki Marine Biol. Stat., Fac. of S@ilo 5
Univ. of Tokyo. Kanagawa. Dept. of Physi-
ol. and “Dept. of Biochem., Dokkyo Univ.
Sch. Med., Tochigi. * Dept. of Mol.Biol.,
Keio Univ. Sch. Med., Tokyo.
Unfertilized eggs of the pacific her-
ring, Clupea palasii, release proteins
which activate motility of herring sperma-
tozoa. We have previously reported the
purification of herring sperm activating
proteins (HSAPs) and revealed that HSAPs
are small acidic proteins on SDS-PAGE
(Ohtake et al., 1990). To analyze the
molecular natures of HSAPs, the HSAPs
obtained from isoelectric focusing column
were further analyzed by immobilized pH
gradient gel electrophoresis. HSAPs are
at least 5 proteins which pI values are
4.8, 4.9, 5.0, 5.1 and 5.4, respectively.
The MW of all HSAPs purified on immobi-
lized pH gradient gel electrophoresis are
almost equal and are estimated less than 7
KD on SDS-PAGE. Gel filtration estimates
the MW of HSAPs about 14 KD, which value
is twice the MW obtained on SDS-PAGE.
These suggest that HSAPs exist as dimers
under the physilogical conditions. SDS-
PAGE also revealed that HSAPs are composed
of at least two peptides having different
MW.
Role of Ca2*-channel in activation of
Xenopus egg by sperm extract.
Y. Iwao, N. Se, and S. Jikumaru.
Biol. Inst., Fae. Sci., Yamaguchi
University, Yamaguchi.
Potential changes as well as voltage-
dependence on fertilization of amphibians
are characteristics of sperm species, so
that we attempted to obtain and
characterize a sperm factor to induce egg
activation. The extract obtained from
Cynops pyrrhogaster sperm induced
activation of dejellied, unfertilized
Xenopus laevis eggs. The eggs treated
with the extract elicited a short-lived,
positive-going potential after appearance
of a deep hyperpolarization. The pattern
of potential changes induced by the sperm
extract was quite similar to that by
Cynops sperm, but not by homologous sperm.
Immature oocytes or fertilized eggs show
no potential changes upon treatment with
the extract. When the unfertilized eggs
were treated with the extract in 340 pM
Ca“, the onset of cortical contraction
and the positive-going potential was
accelerated. The activation was
inhibited by voltage=clanping at higher
than -10 my in 34 pM Ca” , or -20 mV in
340 uM Mg , respectively. The
activation) was not inhibited at +20 mV in
340 pM Ca These results indicate that
the activation by the Spey extract is
mediated by opening of Ca channels on
egg plasma membrane.
HEPARIN INHIBITS CALCIUM TRANSIENTS IN
FERTILIZED SEA URCHIN EGGS INDUCED BY
SPERM AND ITS SOLUBLE EXTRACTS.
M.Osawa ,_H.Uchiyama~, H.kysudas,
N.Kaneko and H.Kuroda“., Sugashima
-B.L.,Sch of Sci., Nagoya Univ. Toba,
Dept. of Biol., Fac of Scaly toyamd
Univ., Toyama.
Fertilization is known to initiate a
transient increase of intracellular calci-
um concentration (Ca;-transient) and an
accompanying change of membrane potential
in sea urchin eggs.
Using aequorine, we previously showed
that sperm and its soluble extract caused
a Ca,-transient in fertilized eggs from
which fertilization membranes and the
hyaline layers were removed.
In this report, we quantitatively meas-
ured intracellular Ca concentration with
Indo-1 microfluorometry and showed that
the microinjection of heparin (final
concentration of approx. 1mg/ml) into
fertilized eggs inhibited the Ca;- and
voltage- transients by sperm and sperm
extract. The sperm extract which was
diluted to 10-fold with artificial sea
water (ASW) increased the intracellular
Ca*+ concentration from 240nM to 760nM (in
average values). The sperm extract which
diluted to 1000-fold lost activity. The
molecular weight of the active factor in
the sperm extract was less than 5000.
Developmental Biology 1207
TPA-SENSITIVE PROTEIN PHOSPHORYLATION INTERFERES WITH
CORTICAL REACTION AND INDUCES CYTOSKELETAL CHANGES IN THE
SEA URCHIN EGG.
A. Morimatsu*, H. Murofushi* and I. Mabuchi*. *Dept.,of
Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo,
Tokyo.,and Department of Biology, Fac. of Arts and
Sciences, Univ. of Tokyo, Tokyo.
When unfertilized sea urchin eggs were treated with TPA
and then inseminated, no cortical reaction occurred and no
fertilization membranes (FMs) appeared. Also calcium
ionophore A23187 did not induce the formation of the FMs
in the TPA treated eggs. These effects were completely
abolished by a C-kinase inhibitor H7. Furthermore,
calyculin A-treated unfertilized eggs did not form the FMs
upon addition of A23187. Numerous bundles of actin
filaments were observed in the TPA-treated eggs.
Next, we added 200nM Ca** to cortices isolated from
unfertilized eggs. The cortical reaction did not occur in
those isolated from the TPA treated eggs, but it did in the
control COrtices and the FMs were formed.
When the TPA-treated eggs were inseminated, they became
polyspermy. They began to divide irregularly at 120-150min
after insemination to produce 4 to 17 cells. At this stage
some eggs did not divide but began to move like amoebae
(Mabuchi, Morimatsu & Tosuji, this meeting). These results
indicate that some DG-dependent kinases are involved in the
cortical reaction and cytoskeletal organization in the sea
urchin eggs.
CHANGES IN INTRACELLULAR FREE CALCIUM
AT FERTILIZATION IN MACTRA CHINENSIS
OOCYTES.
R. Deguchi and K. Osanai. Mar. Biol. Stn.,
Asamushi, Tohoku Univ., Aomori.
Oocytes of Mactra chinensis (Mollusca, Pelecypoda)
are spawned at prophase-| and resume meiosis by
fertilization. The prophase-| arrested oocytes (GV-
oocytes) were injected with fluorescent Ca-indicator
fura-2 to measure changes in intracellular free Ca™
(Ca?*i) at fertilization. Shortly after insemination, Cai
increased rapidly and decreased within 2 min. After
that, Ca*i was retained higher than the resting level (post-
fertilization level) for a while and returned to the resting
level about 10 min following fertilization. Germinal vesicle
breakdown (GVBD) occurred 12-15 min after fertilization.
External Ca* was required to maintain the post-fertilization
level because Ca**i returned to the resting level
immediately after replacing external medium with Ca-free
artificial seawater containing 10 mM EGTA. In order to
induce GVBD, Ca?*i had to be kept above the post-
fertilization level for more than 4-5 min. Oocytes of another
bivalve Limaria hakodatensis didn't require Ca™i increase
to undergo GVBD. In Limaria oocytes, Ca*i level was
higher than the post-fertilization level in Mactra oocytes.
It is probable that Ca?*i level above a threshold is
prerequisite for GVBD in bivalve oocytes.
ULTRASTRUCTURAL STUDIES ON A TISSUE-
SPECIFIC DIFFERENTIATION OF MELANOPHORES
IN AN ORANGE-COLORED VARIANT OF MEDAKA
FISH, ORYZIAS LATIPES.
Eu. Hirose and J. Matsumoto. Department of Biology,
Keio University, Yokohama
In an orange-colored variant of the medaka fish
Oryzias latipes which inherently devoid of the dominant
gene B, melanophores exhibit a tissue-specific
differentiation, as exemplified with failure of melanogenesis
in the skin , an incomplete or deteriorated melanogenesis
in the choroid and the peritoneum and formation of a
mosaicism like melano-iridophores in the peritoneum. Ina
wild strain of this species carrying B gene, all melano-
phores are terminally differentiated irrespective of the
tissues they are located. This indicates that the deficiency
of B gene impairs the differentiation of melanophores in
the medaka. Electron microscopy disclosed that the failure
or deterioration of melanogenesis in fish deficient in B gene
occur inside the melanosomes and that the manner by
which deterioration give rise to is different between
melanophores in the skin and the choroid or the
peritoneum. The ubiquitous occurrence of reflecting
platelet-laden melanophores in the peritoneum of this
variant and the total absence of a mosaicism in pigment
cells of the wild strain indicate that the deficiency of B gene
imposes melanoblasts distributed in this tissue to an
ambiguous state with regard to their differentiation. Little
difference is observed with regard to melanosome
maturation in pigment epithelial cells between orange-
colored variant and the wild strain, indicating an silent role
of the B gene in their differentiation.
THE EXPRESSION OF CELL ADHESION MOLECULES
IN PIGMENT CELLS.
fe Fukuzawa and M. Obika, Dept. of
Biology, Keio Univ., Yokohama, Japan
To study the molecular mechanisms of
pigment pattern formation, we examined the
expression of cell adhesion
molecules(CAMs) in pigment cells, since
little is known about the cell-to-cell
communication in pigment cells. Three
types of pigment cells (melanophores,
xanthophores, and iridophores) were
cultured from the medaka, Oryzias latipes,
and the expression of N-CAM and N-Cadherin
was examined by immunocytochemistry. The
results showed that both N-CAM and N-
Cadherin were expressed in xanthophores,
but not in melanophores and iridophores.
N-CAM staining was observed in the tip or
the base of the dendrite, or in the edge
in-between the dendrites of xanthophores
as a spot. If the shape of xanthophores is
round in the presence of fibronectin and
collagen type I, N-CAM was detected in the
edge as a short line, or in the peripheral
area as a half-ring whose center lay on
the edge. The staining pattern of N-
Cadherin in xanthophores was almost the
same as that of N-CAM, however, the
staining area of N-Cadherin was broader
than that of N-CAM. Furthermore, the
number of stained area of N-Cadherin was
higher than that of N-CAM. In our
knowledge, this is the first demonstration
of CAMs in pigment cells.
1208 Developmental Biology
DETERMINATION OF GENOTYPES IN QUAIL PLUM-
AGE MUTANT (BLACK AT ATCH, Bh) MBRYOS .
Y. Kubotg’, N; Shiojiri', T. Kaneko” and A.
Nakamura“. Dept. Biol., Fac. Sci., Shi-
zuoka Univ., Shizuoka. Dept. Biol.,
Hamamatsu Coll., Univ. Shizuoka, Hamamatsu.
The Bh homozygotes die during early
development (3-7 days), showing subcutane-
ous haemorrhage, and degeneration of liver
and neural tube tissues (Minezawa & Wakasu-—
gi, 1977). No reports have been done on
determination of genotypes of Bh embryos
before feather germ development. We
report the way of determining the genotypes
of young Bh embryos with chorioallantoic
membrane (CAM) grafting of the skin.
Embryos obtained by the cross of Bh/+ x
Bh/+ quails were used. The 7-day thigh
skin fragments in which no feather germs
developed yet were grafted onto the CAM of
chick embryos, and cultured for 9 days.
The grafts developed feather germs with
black and yellow stripes, grey feather
germs, and brown feather germs. The ratio
of the grafts developing the striped, grey
and brown feather germs was 1:2:1. The
results suggest that the grafts of the
striped, grey, and brown feather germs are
derived from +/+, Bh/+ and Bh/Bh embryos,
respectively. A few embryos with brown
feather germs and subcutaneous haemorrhage
which might be homozygous were observed at
10 days, supporting the above idea. In
the 7-day Bh/ Bh embryos, subcutaneous
haemorrhage occurred, but liver and neural
tube degeneration was not always seen.
IN VITRO CLONAL ANALYSIS OF MOUSE NEURAL
CREST DEVELOPMENT.
K.Ito!, T.Morita!, and M.Sieber-Blum?.
Honea, one ihloit,, (ili, Cn’ (en, Jachoe. .
Osaka Univ., Osaka, Dept. of Cell Biol.
and Anat., Med. Coll. of Wisconsin, USA.
We established a clonal culture system
of mouse neural crest cells in order to
analyze developmental potentials of these
cells. Three morphologically distinct
types of ‘clones were observed. (1) "Pig-
mented clones" consisted of melanocytes
only, suggesting that the clone-forming
cells were committed to the melanogenic
lineage. (2) "Mixed clones" consisted of
pigmented and unpigmented cells. The
clones contained up to four types of
cells; melanocytes, S100-positive cells
(Schwann cells or melanogenic precursor
cells), serotonin (5-HT)-positive auto-
nomic neuron-like cells, and substance P
(SP)-immunoreactive sensory neuron-like
cells. Thus, at least some mixed clone-
forming cells are pluripotent. (3) Un
pigmented clones" consisted of unpigmented
cells only. The clones contained up to
three types of cells; S100-positive cells,
5-HT-positive cells, and SP-immunoreactive
cells, Suggesting that some precursor
cells of these clones have partially
restricted developmental potentials.
These results indicate that the mouse
trunk neural crest is a heterogeneous
population of cells with different types
of developmental potentials.
MELANOCYTE DIFFERENTIATION AND C-KIT EXPRES-—
SION IN CULTURED MQUSE NEURAL EST CELLS.
H.Ono?, _ Y.Kawa*, M.Sato*, T1.Baba2,
.Kubota2, M.MizoguchiY~ and T.Takeuchil.
Biol. Inst., Fac. of Sci., Tohoku Univ.
2Dept. Dermatol., St. Marianna Univ. Sch. of
Med.
The signal transduction system by c-kit and
SLF (Steel factor) affects development of
melanocytes. In this study, we attempted to
detect the c-kit expression in neural crest
cells (NCCs) by monoclonal antibody, ACK2,
and compare it with the pattern of melano-
cyte differentiation in vitro. NCCSs were
induced to differentiate into mature melano-
cytes by addition of 10 ng/ml PMA (phorbol
12-myristate 13-acetate) into the culture
medium. Further addition of 10 nM cholera
toxin resulted in an increase in the number
of melanocytes. Melanocyte differentiation
seems to be correlated with the c-kit ex-
pression in NCCs. The c-kit positive cells
were more than 1000 cells/explant when
treated with PMA and cholera toxin, and
about 500 cells with PMA only by 15 days in
culture. The number was less than 100 cells
without PMA. (Total number of NCCs were not
affected by the culture media.) We assume
that PMA induces c-kit expression and that
cholera toxin supports this action. Subse-
quently, the c-kit/SLF signal transduction
system accelerates the differentiation into
melanocytes. The c-kit positive NCCs formed
clusters in explant at 9 days in culture,
this indicates that NCCs are heterogeneous
and that some of NCCs have lost ability of
c-kit expression by this stage.
KERATINOCYTES REGULATE THE DEVELOPMENTAL
CHANGES OF THE PROLIFERATIVE ACTIVITY OF
MOUSE EPIDERMAL MELANOCYTES IN SERUM-FREE
CULTURE. .
T. Hirobe. Div. of Biol., Natl. ainsteusot
Radwvols (Scie, Chibas
The proliferative activity of mouse
epidermal melanocytes is known to change
during development. In order to make clear
the control mechanism of melanocyte proli-
feration, epidermal cell suspensions from
mice of various ages were cultured with
serum-free medium containing dibutyryl
cyclic AMP and basic fibroblast growth
factor. When the epidermal cell suspensions
of dorsal skins of 0.5-, 2.5- and 4.5-day-
old C57BL/10J mice were cultured on poly-
styrene dish, melanoblasts proliferated
well around keratinocyte colonies. In
contrast, when the epidermal cell suspen-
sions of 7.5-, 20.5- and 60.5-day-old mice
were cultured similarly, keratinocytes
failed to attachto the dish and melanoblasts
did not proliferate. However, when the
epidermal cell suspensions of 7.5-, 20.5-
and 60.5-day-old mice were cultured on
Type I collagen-coated dish, keratinocytes
attached to the dish and melanoblasts pro-
leferated around the keratinocyte colonies.
These results suggest that keratinocytes
are involved in regulating the prolifera-
tion of mouse epidermal melanocytes and
that the developmental changes in the pro-
liferative activity of epidermal melano-
cytes are caused by the changes in the cell
adhesion property of keratinocytes.
Developmental Biology 1209
CLEAVAGE ASYNCHRONY IN THE TUBIFEX EMBRYO:
INVOLVEMENT OF CYTOPLASMIC AND NUCLEAR
FACTORS
fT. Shimizu, Zool. Inst., Fac. of Sci.,
Hokkaido University, Sapporo 060
AB cell of the 2-cell embryo of
Tubifex divides 40 min later than CD cell.
A previous study has shown that AB cell
enters M-phase 5 min later, and spend in
prometaphase portion of M-phase about 35
min longer than CD cell. In the present
study, nuclear behavior was examined in
embryos where first cleavage furrows were
regressed or blastomeres were electrically
fused. When AB and CD nuclei were brought
into a common cytoplasm, they entered M-
phase simultaneously. The durations of M-
phase portions from prometaphase through
telophase were not altered even in a
common cytoplasm, as long as mitotic
figures of AB and CD nuclei were separated
from each other. Only when the mitotic
spindles of both nuclei were united at
poles, the length of prometaphase of AB
nucleus was significantly shortened, and
phases of the chromosome cycle became
synchronous. Mitotic spindles are
morphologically distinct between AB and CD
cells of intact embryos; morphology of the
spindle was not altered by changes in
cytoplasmic environment. These results
suggest that cleavage asynchrony in the
Tubifex embryo is brought about not only
by cytoplasmic components but also by
nucleus-associated factors.
PRODUCTION OF GERM LINE CHIMERAS IN
MEDAKA.
Y.Wakamatsu', M.Kinoshita?, H.Toyohara?,
M.Sakaguchi?, T.Iwamatsu*, Y.Taguchi‘, H.
Tomita®, K.Ozato'. 'Fac. of Lib. Arts and
Sci., and ?Fac. of Agri., Kyoto Univ.,
Kyoto, *Dept. of Biol., Aichi Univ. of
Edu., Kariya, ‘Inst. of Radiol. Sci.,
Chiba, ®*Fac.of Sci., Nagoya Univ., Nagoya.
To produce germ line chimeras in
medaka, cells of morula or blastula
embryos of an inbred strain of wild type
(HNI-I) were taken out and injected into
about 200 embryos at the same stages of
inbred (HO5) and outbred strains of an
Orange-red variety and two strains of
albinos. In some experiments, dechori-
Oonated embryos were used. After injection,
embryos were cultured and bred at 26°C.
Black-pigmented cells developed in yolk
sacks, pigment epithelia of eyes, skins,
fins, and peritonea. Until now, 26 orange-
red individuals have sexually matured.
They were mated with the same strain.
About 10% of F, progeny from a male
chimera exhibited the wild type body
color. Isozyme analyses showed that these
wild type F, were hybrids of the donor
and recipient strains. When the wild type
F, were backcrossed to the recipient
strain, phenotypes in the body color in
BC, segregated into the wild type and
orange-red at a 1:1 ratio.
MORPHOGENESIS OF MEDAKA EMBRYO (FORMA-
TION OF EMBRYONIC AXIS)
Y. Ebina and S.Yokoya Div. Cell Sci.
Fukushima Med. Col. Fukushima
In the medaka embryo, shortly after gas-
trulation begins, cell convergence occurs
and the embryonic axis appears at one side
of blastodisk (the dorsal side) where the
embryonic shield is formed. The mechanisms
involved in the formation of the embryonic
axis in the teleost are not well under-
stood.
We examined the cell morphology in the
prospective embryonic shield region in the
medaka embryo during epiboly and gastrula-
tion with scanning electron microscopy.
SEM revealed that the marginal cells of the
enveloping layer in the prospective embryo-
nic shield region at late blastula are
different in shape and in contact relation
with inner yolk syncytial layer from the
Marginal cells in the other region.
These differences may cause cell conver-
gence.
1210 Genetics
KARYOTYPES AND BANDING PATTERNS IN EIGHT
SPECIES OF THE SCORPLONFISH(SCORPAENIDAE).
T. Yokoyama’, N.Ebitanivand T.Kubo!. Biol.
Lab., Sophia Univ., Tokyo and *Biol. Lab.,
Shohoku Coll., Atsugi.
Somatic chromosomes were prepared from
the cultured cells derived from fins of
fishes. The chromosome numbers in eight
species of scorpionfishes(Helicolenus
hilgendorfi, Scorpaena neglecta miostoma,
S.izensis, S,neglecta neglecta, Sebastes
inermis, S.matsubarae, Sebastiscus albo-
fasciatus and S.marmoratus) were all 48.
Karyotypes in these species, in the order
of the species mentioned above, were as
follows: 2M+2SM+44A, 8M+16SM+34ST, 8M+24ST
+16A, 6M+2SM+30ST+10A, 2M+46A, 2M+46A, 4M
+2ST+42A and 2M+46A. One pair of AgNORs
were found on short arms of metacentrics
in the three species of genus Scorpaena
and on acrocentrics in S.matsubarae and
S.albofasciatus. In the other three spe-
cies, two pairs of silver-stained bands
were revealed on two pairs of acrocentrics.
C-bands were found on one pair of subtelo-
centrics in the fishes of genus Scorpaena.
In the other five species, they were found
on more than eight pairs of acrocentrics.
Based on these findings,relation between
the characteristics of chromosomes and
diversification of species in the scorpion-
fishes was discussed.
CELL CULTURE AND CHROMOSOME ANALYSES OF
XENOPUS BOREALIS.
K. Sekiya Dept. of Biol., Fac. of Sci.,
Niigata Univ., Niigata
Cell nuclei of Xenopus leavis are
stained homogeneously by fluorescent dye
quinacrine, whereas those of X. borealis,
which are able to get interspecies hybrids
with xX. 1., are stained with about 15
bright fluorecent spots. As a result of
staining cell nuclei of X.b. by quinacrine
mustard, acridine orange and DAPI, bright
spots could be observed in the various
tissues. Especially DAPI and quinacrine
mustard staining is clearly visible.
X. b. whole embryos (st. 44) were used
for setting up the culture, and in the
primary culture, the surface of the bottle
was covered with some different-type
cells. The floating cells, selected by
light pipetting, could be kept more than 3
months. In the cells attached to the
surface, their fluorescent spots tend to
become unclear and decrease, but the spots
are kept even in the 15th subculture.
The chromosome of both species is
Similar, but their different type of sat-
chromosome could be confirmed in silver
staining.
The results obtained in these cell
culture will be very useful for
interspecies cell fusion.
ANALYSIS OF MALE CHROMOSOMES FROM THE
HYBRID ANDROMEROGONES OBTAINED BY USING
CRYOPRESERVED SPERM OF SEA URCHINS.
K. Saotome!, R.Kamimura2, S.Kurokura? and R.
Hirano. !Yokohama City Institute of Health,
Yokohama, 2Kitazato Univ., Iwate and 3Univ.
of Tokyo, Shizuoka.
Analysis of male chromosomes is pos-
sible by the use of hybrid andromerogones
obtained by inseminating non-nucleate egg
fragments of Hemicentrotus pulcherrimus with
sperm of different species (Saotome, 1991).
This system, however, was not applicable to
the species having different breeding season
from that of_H. pulcherrimus. Cryopreserved
sperm was used to dissolve this problem. The
freeze-thaw procedure of sperm was carried
out as follows:1l)dry sperm was diluted with
sea water containing 10% DMSO at the con-
centration of 2-10%, 2) the suspension was
pipetted into plastic insemination straws,
heat sealed and frozen gradually to -196°C,
3) straws were stored in liquid nitrogen and
4) frozen samples were thawed rapidly by
immersing the straws in sea water at room
temperature. The sperm of five speceis was
used;Strongylocentrotus nudus, Pseudo
centrotus depressus, Strongylocentrotus
intermedius, Glyptocidaris crenularis and
H. pulcherrimus. Active swimming of sperma—
tozoa was observed after thawing. The rate
of cleavage varied from 6 to 84% according
to species or batch of egg fragments and
cryopreserved sperm. Chromosome numbers were
haploid of those already reported in five
species and karyotype of their chromosomes
could be analyzed.
CHROMOSOME MOVEMENT DURING FIRST POLAR
BODY FORMATION IN THE ISOLATED MEIOTIC
APPARATUS OF TUBIFEX HATTAI (OLIGOCHAETA,
TUBIFICIDAE)
M-. Matsumoto. pept. of RBiol., Fac. of
sci., yamagata yniv., yamagata
yolky eggs of the freshwater oligochaete
j~. nattai were freed from their cocoons
in Lehmannts solution. The vitelline mem-
brane and egg cortex of the fresh eggs
were ruptured and yolk granules were dis-
persed in a petri dish containing 2y
glycerol. The opaque core of the egg
gradually emerging within 2-3 minutes of
the dispersion was transferred to isolation
medium. These meiotic apparatus were glued
on a slide glass, and stained. puring
anaphase J, formation of the cleavage
furrow of tne polar body began during
migration of the half-bivalent into the
polar body when the temperature was
changed. Furthermore, the separation of
the first polar body from the egg was
completed before the termination of chro-
mosome segregation. That is, some cnhromo-
somes that should have migrated into the
polar body remained in the egg cell. These
phenomena were found 34 of the cells with
a temperature shift of 20¢—+26¢, but 294%
with a shift of 26¢—23%. when meiotic
apparatus were taken from eggs with shifts
of 19¢—922% and 22¢-+17%, we found a
complete polar body.
Genetics
EFFECTS OF MICROWAVE ON GERM CELLS OF
DROSOPHILA.
K.Kimura-. tokyo
Hokkaido
Y.Tonomura? and
Womans ' Christ. Univ. and
Inst. Tech.
The Study aimed at finding the
effects on germ cells. 1) Specimens
used were 72-hour old D.melanogaster,
late third-instar larvae, young pupae
and late pupae with microwave appli-
cation time of 5 seconds, 10 seconds
and 30 seconds using microwave of 2.45
GHz at 200W, to see the mechanism of
cell division and the protein synthesis
of adult flies. 2)In this study,
cytological specimens were made by
air-dry method and the primary electro-
phoresis method were used for the bio-
logical analysis of protein synthesis
of sibling cells. 3)We are currently
conducting an observation using more
than 690 microphotograph of germ cells
of 20 females and 20 males taken from
each group exposed to microwave at
varied duration (excluding late pupae)
to see whether or not cell division has
taken place. An electrophoresis study
disclosed no differences in the effects
of microwave irradiation on larvae
while some differences were observed on
pupae, which may suggest the loss of
protein bands in the molecular-weight
region of 100,000.
A GROUP OF CAFFEINE HYPER-SENSITIVE
MUTANTS IS HIGHLY MUTABLE.
T.Kusano!, Y.Wataya2, K.Okada3 and
H, Shinkawa4.
‘Hiroshima women's Univ. 20kayama Univ.
Hiroshima Univ. Hospital and 4Hiroshima
Univ.of Economics.
Caffeine is a clastogen but not likely
mutagen to mammalian cells in culture.
To catch any clues of much unknown effects
of caffeine to cells,we isolated caffeine
sensitive'cellfrom mouse FM3A cell line.
So far, we could identified two groups.
First group which Fcaf22B (C2)belongs to
had not shown hyper-mutability. In second
group Feaf(NQ@) 1 (NQA), which was selected
in diluted caffeine showed hyper-mutability
and hyper-sensitivity to bromodeoxuridine.
FBrdUrd 1 (A2) was another caffeine sensi-
tive mutant because this was selected first
as a bromodeoxyuridine sensitive mutant.
A2 mutant too found hyper mutable,but hyp-
er sensitivity decreased in the presence of
deoxycytidine. These results caused douts
if caffeine could have potential mutagenic
activity as bromodeoxyuridine has.
Hyper mutability of both cell lines was
seen frequently but mutation rate was not
likely to differ from parent FM3A .
dNTP pool size were mesured and in NQA cell
an unbalnce was observed. Unbalnce of dNTP
could occur frequently and spontaneou fre-
quent mutation in NQA and A2 mutants might
corelate to this phenomenon.
Caffeine is still an atractive mutagen.
1211
POPULATION STRUCTURE OF ALLELES FOR THE
PLASMODIUM FALCIPARUM MEROZOITE SURFACE
PROTEIN.
So Jongwutiwes’, f° Nakazawal, H. Kanbara
and K. Tanabe~. Div. of Protozool.,
Inst. of Trop, Med., Nagasaki Univ.,
Nagasaki and “Lab. of Biol., Osaka Inst.
of Technol., Osaka.
The precursor to the P. falciparum
major merozoite surface protein (MSP1) is
one of the potential malaria vaccine can-
didates. Our previous studies have
demonstrated that the MSPl gene of
laboratory strains of P. falciparum ex-
hibits allelic variation. Here, we ex-
amined the extent of the allelic variation
in natural isolates obtained from an en-
demic area of malaria by Southern blot
hybridization. DNA fragment probes and
oligonucleotide probes were derived from
variable blocks of two allelic forms of
the MSPl gene. Hybridizations against
genomic DNA of 18 isolates obtained from
Mae Sod district in Thailand revealed the
existence of 7 MSPl alleles, 2 of which
were newly identified. Coexistence of
different alleles in individuals was not
rare. It was also noted that the 7 al-
leles did not occur at the same frequency,
but rather several alleles predominated in
the population of P. falciparum in the
malaria field analysed.
1
DISCRIMINATION AMONG APHID CLONES BY
ARBITRARILY PRIMED POLYMERASE CHAIN
REACTION (AP-PCR).
T. Fukatsu and H. Ishikawa.
Zool. Inst., Fac. Sci., Tokyo Univ., Tokyo.
When genomic DNA is amplified by PCR with a
primer of an aibitrary sequence in low stringency and
electrophoresed, ladder—pattern specific to the genome
used will be obtained. This method, AP—PCR, seems to
be superior to the conventional DNA fingerprinting in
the following aspects; 1) universally applicable to any
organism's genomic DNA, 2) only very small amount of
template DNA is needed and 3) can be done without
blotting or hybridization procedures. We applied this
method to discrimination among aphid clones.
We adopted a 15mer primer, S'atgcaggaptcgcat3',
in this study. When DNAs from various organisms such
as aphids, moth, fish and human, were amplified with
this primer, species—specific ladder patterns were ob—
tained in all of them. To establish optimal reaction
condition and to confirm reproducibility, the genomic
DNA from a laboratory—maintained clone of Acyrthosi—
phon pisum was subjected to AP=PCR. Through a wide
range of template DNA amount, from ae 800ng,
identical ladder—patterns were obtained. he patterns
were also identical irrespective of aphid's instar. The
patterns did not change through five: parthenogenetic
generations. 2 : p
Ceratovacuna nekoashi is an aphid which forms
banana-like galls on storax. A gall is composed of more
than 10 subgalls, and all the insects in a gall are parthe—
nogenetic offsprings of a single fundatrix. When AP—
PCR was applied to this aphid species from several galls
on the same storax twig, the epraules were, as expected,
identical between subgalls of the single gall but distin—
gushable between different galls.
1212
MACRONUCLEAR ANLAGEN INDUCES PROLONGED
BACKWARD SWIMMING IN NONEXCITABLE MUTANTS OF
PARAMECIUM, CNRS, JUST AFTER TRANSPLANTATION.
N. Haga. Dept. of Biotech., Senshu Univ. of Ishinomaki,
Ishinomaki.
Paramecium has two types of nuclei, micro- and
macronucleus, in the cell. These nuclei are same in
developmental origin but different in size, DNA content
and function. During macronuclear development,
reorganization of chromosomal DNA including gene
amplifications and rearrangements has been done.
However the molecular mechanisms of nuclear
differentiation is still unknown.
| found that when macronuclear anlagen was
transplanted into a vegetative cell of wild type in
behavior, the recipient cell showed backward swimming
for about 60 sec in the K-Dryl's solution. Physiological
and electrophysiological studies have demonstrated that
ciliary reversal is induced by the increase of
intracellular calcium ion concentration. Since the
control injection of vegetative macronucleus into a wild
type cell induces short backward swimming for about
10 sec, nonexcitable mutants, CNRs, were used as
recipients. Both cnrA and cnrB cells showed backward
swimming for 50-60 sec when macronuclear anlagen
was injected but did not swim to back when vegetative
macronucleus was injected. These results suggested at
least two possibilities, one is that Calcium ion is
enriched in macronuclear anlagen and the other is
anlagen induces the increase in intracellular Calcium ion.
PHENOTIPIC CHANGE IN SYNGEN SPECIFICITY OF MATING
TYPE BY INJECTION OF MACRONUCLEAR NUCLEOPLASM IN
Paramecium caudatum.
M. Hori and M. Takahashi. Inst. Biol. Sci., Univ. of Tsukuba, Ibaraki
Paramecium caudatum has sixteen sub-groups called
'syngen' which consists of complementary mating types (E and O
mating types). From cross breeding analysis, Tsukii(1985) showed
that syngenic specificity are controlled by three loci: E type is
controlled by codominant alleles at the Mt locus [e.g. E type of
syngen 3 (E°) is controlled by Mt*] , O type by MA and MB.
To get more information about the relationship between
syngens, transplantation of nucleoplasm of macronucleus was
performed between two syngens by microinjection using the cells
immobilized by partial decilliation with 5% ethanol.
In intra-syngen transplantation, recessive phenotypes in
behavior and exocytosis (cnrB and tnd2 ) were rescued by the
nucleoplasm of wild type and O mating type also was turned to E
type by injection of E type nucleoplasm.
On the other hand, in inter-syngen transplantation between
syngen 3 and syngen 12, although both cnrBand tnd2 were
rescued by dominant genes over syngen, the mating types,
however, changed their syngen specificity. That is, when
nucleoplasm of E'? was injected into macronucleus of O°, 11 out of
43 clones established expressed E” in spite of containing no Mt°
allele. It was also true in the reciprocal combination, E® to O'*. This
result suggests that syngenic specificity of Mt gene converted that
of donor into recipient by inter-syngen transplantation.
Genetics
A MICRONUCLEUS-SPECIFIC SEQUENCE EXISTS IN
5’-UPSTREAM REGION OF CALMODULIN GENE IN
TETRAHYMENA THERMOPHILA.
M.Katoh, M.Hirono!, T.Takemasa, M.Kimura
and Y.Watanabe. Inst. of Biol. Sci., Univ.
of Tsukuba, Tsukuba and !Cell Bank, Inst.
of Phys. and Chem. Res. (RIKEN),
Tsukuba.
To know relationships between DNA
rearrangement and switching of gene ex-
pression during macronuclear development
in Tetrahymena thermophila, we analyzed
micro- and macronuclear genomic DNAs for
calmodulin gene by Southern hybridization
and sequencing. Here we presented evidence
that a 1.4 kb micronucleus (mic)- specific
sequence existed about 3.5 kb upstream
from the initiation codon of calmodulin
gene, suggesting the occurrence of DNA
rearrangement. The mic-specific sequence
was highly AT-rich (85%) and had ATTA at
both ends. The mic-specific sequence
included a T-rich tract, Ti6CTs5, in the
middle region and nearly complementary A-
rich tract, As5TA10GAs, existed at 7 bp
upstream of initiation codon. In addition,
20 bp repetitive sequences TAAT(TAAC) 4
were found about 100 bp upstream of the
mic-specific sequence and in the promoter
region of calmodulin gene (about 120 bp
upstream of initiation codon).
Although Significance of 1.4 kb
mic-specific sequence remains unclear at
the present, the TAAT(TAAC)4 and mic-—
specific sequence including TisCTs might
exert an influence upon the conformation
of 5’-flanking region and the transcrip-—
tion of calmodulin gene.
IN VITRO ANALYSIS OF THE PROMOTER REGION OF
TETRAHYMENA ACTIN GENE.
M. Kimura’, M. Hirono“, T. Takemasa! and Y.
Watanabe-. “Inst. of Biol. Sci., Univ. of
Tsukuba, Tsukuba and “Cell Bank, RIKEN Inst. of
Phys. and Chem. Res., Tsukuba
OE EE ———————————————e
In ciliates, little is known about the
promoter elements and their protein factors in a
certain mRNA transcription. Here we demonstrated
fourteen protein binding sites (I-XIV) in the 5’-
flanking region of Tetrahymena actin gene by
using gel retardation assay and DNase I
footprinting. Some of these sites are homologous
to the conserved promoter elements, such as TATA
box (I), GC box(II), GATA box (III and VI),
homeobox protein binding site (IV), TRE (V), and
CCAAT box (VII and IX). The other sites are
little homologous to the previously known
promoter elements. We further investigated the
roles of such protein binding sites in
Tetrahymena actin gene transcription by using in
vitro transcription assay. The binding sites I,
II, III, IV, V, X, and XI activated the
transcription of Tetrahymena actin gene, whereas
the binding sites VI, VII, VIII, and IX repressed
the transcription under the conditions where
nuclear extract from exponentially growing cells
was used. The present results suggest that these
elements (protein binding sites) regulate the
transcription of Tetrahymena actin gene through
interactions with nuclear protein factors.
Genetics
IDENTIFICATION OF HOMEOBOX-CONTAINING
GENES IN THE MOST PRIMITIVE METAZOA,
SPONGE.
M.Seimiya+ , Y.Watanabe!, and Y.
Kurosawa“, ‘4pDept. of Biol., Fac. of
Sci., Ochanomizu Univ., Tokyo, 2Inst.
for Comprehensive Med. Sci., Fujita
Health Univ., Aichi.
Porifera is the most primitive
phylum of metazoa. Genomic DNA from
freshwater sponge (Ephydatia fluviatilis
) was subjected to amplification by the
polymerase chain reaction using two
primers corresponding to helix 1 and
helix 3 regions in the homeodomain.
Sequence analysis of the amplified
products revealed presence of two kinds
of homeobox-containing genes, designated
prox-1 and prox-2, in sponge. The amino
acid sequences of the homeodomains of
prox-1 and prox-2 showed 72 and 62%
identities with those of NK-3 and om of
Drosophila, respectively.These results
indicated that when metazoa appeared
during the course of evolution, multiple
and distinct classes of homeobox-
containing genes identified in higher
organisms had already existed.
CLONING OF 5'REGION OF TYROSINASE GENE IN
ASCIDIANS AND LAMPLEYS.
H.Masuya,H.Yamamoto,and T.Takeuchi
Biol.Inst.,Tohoku Univ.
Tyrosinase, the key enzyme of melanin
bio synthesis, functions in various organ-
isms. In vertebrates,tyrosinase gene ex-
pression is restricted in pigment cells.
In order to elucidate evolution of this
tissue spesific expression, we have cloned
and compared tyrosinase genes from various
vertebrates including human, mouse, Japa-
nese quail, and snapping turtle.
In this study, we carried out cloning of
tyrosinase gene in ascidian, Halocynthia
roretzi, and agnathian, Entosphenus japon-
ics.
In the gene cloning in ascidian, 10kb of
genomic fragment involving 590 base region
that has homology to the first exon of
mouse tyrosinase gene (60% DNA, 48% amino
acid identities) was obtained by screening
genomic library with mouse tyrosinase cDNA
(Tyrs-J). In putative cupper binding reg-
sion 1, 14/19 (73.6%) amino acid identities
and two Hiss those are probabie cupper
binding sites were conserved.
In lampley, a partial library was con-
structed of DNA extracted from a portion of
gel where Southern blot signal was detect-
ed. 4 clones were obtained by screening the
library.
1213
PRODUCTION OF TRANSGENIC ORANGE-
COLORED VARIANTS OF MEDAKA BEARING
MOUSE TYROSINASE GENE BY MEANS OF
ELECTROPORATION AND EXPRESSION OF WILD
TYPE PIGMENTATION IN THEIR SKIN
J.Matsumoto', E.Hirose’, K.Miyazaki’, H.Yamamoto? and
T.Takeuchi? ‘Dept. of Biol., Keio Univ., Yokohama, *Biol.
Inst., Fac. of Sci., Tohoku Univ., Sendai.
We introduced reconstructed mouse tyrosinase
gene, mg-Tyrs-d, into fertilized eggs of an orange-colored
variant of medaka Oryzias latipes by means of
electroporation. Of 589 eggs treated, 38 fish (6%)
exhibited brownish skin pigmentation which was variable
individually but discernible from the untreated. Piercing the
chorion with a tungsten needle before electroporation
markedly increased the yield of individuals with brown
Pigmentation. Crossing between transgenic founders
thus obtained yielded offsprings with heavily brown
pigmentation. Light and electron microscopy disclosed
that melanization was occurring restrictedly in particular
dendritic or disc-shaped cells which were presumed to
correspond to "amelanotic” melanophores present in an
orange-colored variant of this species. The melanosomes
found in such melanophores were peculiar with respect to
their internal structure and different from those in
melanophores of the wild medaka. Immunohistochemistry
using the antibody raised against mouse tyrosinase
disclosed that these melanized cells were specifically
labelled, indicating the expression and transmission of the
gene introduced in transgenic medaka.
FUNCTION AND STRUCTURE OF THE AGOUTI LOCUS
IN THE MOUSE
y.Sato and T.Takeuchi .Biological Insti-
tute, Tohoku University, Aoba-yama, Sendai
980 Japan
The agouti locus( a_) on chromosome 2
encodes the gene product involved in hair
pigmentation. Especially,it determines
type of melanin synthesised in hair-bulb
melanocytes. However,this gene expresses
not in melanocytes but in cells surrunding
melanicytes indicating that an intercellular
communication gives rise to this phenomenon.
In our previous studies,a polyclonal
antibody against skin of the lethal yellow
mouse(A_” /a) was prepared. Some lethal
yellow mouse specific CDNA were cloned by
screening lethal yellow mouse skin cDNA
library with this antibody ( Sato et al.,
1988).
In order to verify the specificity of
these clones, Southern blot analysis of
genomic DNA obtained from several congenic
mice concerning a-locus was performed ,using
one of the clones as a probe. When
genomic DNA was digested with BamHI and
ECORI,Specific bands were detected. There-
fore,it seemes possible that the clone rep-
resents the A ” allele.
1214
RS-2 AND MO MUTANTS IN THE MEDAKA (ORY ZIAS
LATIPES) .
H. Tomita, Lab. of Freshwater Fish
Stocks. Fac. of Sci., Nagoya Univ.,
Nagoya.
The rs-2 mutant(small scales) were
found twice. At the first case, the
rs-2 mutant showed pleiotorpism, that is,
(1) small scales, (2) deformed fin fold
(membrane fin) at larval stages and (3)
inhibition of melanin formation. They
were weak and maintenance of them was
difficult. At the second case, the
rs-2 mutant showed small scales only.
They were kept easily. The F, progeny
of crosses between the first and second
rs-2 mutants were small scales. In the
first rs-2 mutant, (1), (2) and (3) char-
acters might link with each other.
The mo mutant caused pleiotorpic char-
acters, that is, (1) dilution of black
color of melanophores(dilute brown), (2)
disappearance of leucophores at adult
fish, and (3) reduced deposition of gua-
nin on eye balls at larval stages. The
gu-3 mutant resembled to (3) of mo mutant
in a phenotype. The F. progeny of
crosses between gu-3 and mo mutants
caused reduced deposition of guanin on
eye balls. The pleiotorpic characters
of mo mutant consisted of (1) and (2)
characters. The mo mutant might link
with the gu-3 mutant.
THE BASIC BODY WEIGHT OF JAPANESE QUAIL
(Cuturnix cuturnix japonica) FOR BOTH
SEXES
A.Nakamura!, T.Kaneko! and K-.Nishimura?
1 Univ. of Shizuoka, Hamamatsu Col.,
Hamamatsu. 2? Dept.of Biol., Keio Univ.,
Yokohama.
It is usually considered that’ the
weight of the female quail body is heavier
than that of males throughout their devel-
opment and adult stage.
However, as for the growth curve of
pure strain quails,there was no difference
between male and female. The growth curve
difference is due to dimorphism,rapid de-
velopment and slow gene development. Com-
mercial quail lines are caused by contami-
nation of both these genes.
The hens during breeding season were
temporarily heavier, but the body weight
of adult hens and migrating wild quail
hens was equal to that of males during the
nonbreeding season. Domestic commercial
hens were always in an artificial breeding
season.
It is considered that the difference
in commercial quail body weight between
male and female is caused by dimorphism of
the sex-linked semi-dominant genes, moreo-
ver,commercial quail hens are always in an
artificial breeding season. The basic body
weight of Japanese quails is the same _ for
both sexes.
Genetics
ESTABLISHMENT AND SEVERAL PROPERTIES OF NEW
INBRED LINES DERIVED FROM THE JAPANESE
HOUSE MOUSE, Mus musculus molossinus.
Takashi A. Nomaguchi and Youko Sakurai.
Department of Cell Biology, Tokyo Metropol -
itan Institute of Gerontology, Tokyo.
a PT a SS
Inbreedings of 8 lines in molossinus
mouse were started from (Miura ? x Urawa ¢)
F1(MU) in the stock captured at the Kanto
district in Japan and performed by each
primiparous littermate under a clean con-
ventional condition. The respective number
of inbreeding generations of MU/g, MU/a and
MU/£ now exceeds 22, 21, 20, respectively.
Inbreedings of MU/d and MU/e reached to F18
at present, but there were extinct at F11
for MU/h, F9 for MU/b and MU/c. Time
required for inbreeding from F1 to F20 was
BAST AO 3 NASoiW Cles7So Generation time
expressed as days between birth and
primiparous dates of the female in each
successive generation was 117.7 + 50.5
days. It is characteristic for the mouse
to be very small in body weight; the body
weights linearly increased from neonatal
period to 1 month and also further grew
with age, but were constant after 7 months
(19.9 + 1.6 g) for male and 9 months(19.3 +
0.7 g) for female. Maximum body weight
was 21.5 g for male and 20.1 g for female.
Genotypes of Es-1, Es-2, Es-3, Gpd-1, Hbb,
Id-1 and Mod-1 in the 5 lines were
determined and compared with strains of the
laboratory mouse: MU/a for C3H/He type,
MU/d and MU/f£ for C57BL/6, MU/e for
C57BR/cd and MU/g for DBA/2, respectively.
Physiology 1215
TWO KINDS OF FILAMENTS IN THE SMOOTH MUSCLE
CELLS IN THE ADDUCTOR OF A PECTEN, CHLAMYS
NOBILIS.
A.Matsuno, H.Hori and O.Yamada, Dept. of
Biol., Fac. of Sci., Shimane Uni., Matsue
The opaque portion of the adductor of a
pecten was investigated ultrastructurally.
The portion contained smooth muscle cells
which are composed of thick(myosin) and
yhin(actin) filaments. It is well known
that thick filaments in molluscan smooth
muscle cells are composed of a paramyosin
core and myosin molecules coarted the core.
The core shows a regular periodicity.
Thick filaments in the pecten adductor
were classified into two kinds, thinner and
thicker, according to the statistical anal-
ysis of their diameter accumulated from
cross sections. They were correspondingly
classified into two kinds, shorter and
longer, according to the statistical anal-
ysis of their length from isolated native
filaments. These thick filaments were con-
sequently classified into two kinds;
thinner and shorter filaments(about 26.5nm
in diameter and 7.5\m in length), and
thicker and longer ones(about 42.0nm in
diameter and 13.0\m in length).
A regular periodicity appeared on the
surface of each kind of filaments, when
coarted myosin was removed from the sur-
face. Intervals of the periodicity were
Similar in each kind of filament. It is
still obscure whether paramyosin molecules
are differnt each other in the two kinds of
thick filaments, or not.
RESIDUAL FORCE ENHANCEMENT AFTER STRETCH IN FROG
SINGLE MUSCLE FIBRES.
T. Tsuchiya. Department of Physiology, School of
Medicine, Teikyo University, Itabashi-ku, Tokyo.
Stretch of active muscle at a slow velocity
causes force to increase above the isometric level
and this force is composed of, at least, two
component forces, high and quickly decaying one
and low and long lasting one; the latter being
called the residual force enhancement after
stretch. More than one mechanisms are known to be
involved in the former high force enhancement
early after stretch (Sugi and Tsuchiya, 1988;
Amemiya et al. 1988) but the mechanism of the
latter is not well known and investigated in the
present study. A living single skeletal muscle,
tibialis anterior, of the frog was stretched
during tetanus in the low temperature (2-3 C). The
residual force above isometric force 4 or 5s after
stretch was constant irrespective of stretch
velocity and change of velocity during stretch if
the stretch amplitude was constant. The residual
force was higher at longer sarcomere length in the
range between 2.0-3.0um. The ratio of residual
force to isometric force was very much constant at
low and high temperature. Hypertonicity by 98mM
Sucrose had no effects on this ratio of the
residual force. The possibility that the passive
elastic element parallel to cross-bridges was
involved in this phenomenon was discussed.
EXCHANGEABILITY OF F-ACTIN-BOUND NUCLEO-
TIDE IN THE SLIDING MOVEMENT.
N. Oishi Radioisotope Research Center,
Sch. of Med., Teikyo Univ., Tokyo.
It has been shown that F-actins can
change their conformations. A typical
conformation is the rigid form, in which
nucleotides are firmly bound, and the
other is the flexible form that feasibly
exchange the bound-nucleotides. To study
of the structural change in actin during
the sliding movement, the exchangeability
of the actin-bound nucleotide was investi-
gated in an in vitro system.
F-actin containing 3H-ADP was prepared
from rabbit muscle, and the released 3H-
ADP was measured at 30°C. In a nitro-
cellulose-coated microchamber with heavy
meromyosin(HMM), the rate of exchange of
F-actin-bound nucleotide was similar in
the conditions of either sliding(with 1m™
ATP) or rigor(with 1lmM ADP). In the case
of HMM treated with N,N'-p-phenylenedi-
maleimide (pPDM), which showed no ATPase
activity and weak affinity for F-actins,
F-actins were non-motile, but the rate of
exchange of the bound-3H-ADP was almost
the same again. In the presence of both
of the pPDM-treated HMM and the untreated-
HMM,the sliding of the F-actins were
hindered and slowed, and the rate of the
nucleotide-exchange was greatly enhanced.
These data suggest that the load or
tension may transform the actin structure
and facilitate the exchange of the bound
nucleotide.
The Properties of Scallop and Rabbit Striated Muscle
Thin Filaments. Y. Yazawa and M.Kamidochi. Dept. of
Nutritional Physiol., Hokkaido Univ. of Education at
Asahikawa. Asahikawa.
eraction is regulated by troponin-tropomyosin system de-
pendent system dependent on the Ca?' concentration. In
contrast, (a** regulation of molluscan actomyosin ATPase
is known to be associated with the myosin molecule and
myosin-linked system had been regarded as essential.
Recently, we have detected troponin-like proteims
in the striated muscle of scallop
In the present study, we prepared thin filaments from
scallop striated muscle and rabbit skeletal muscle. When
scallop thin filaments were added to scallop myosin, the
Mg**-APTase activity was more deeply inhibited in the
presence of 10°’M Ca** and further activated in the pr-
esence of 10 ‘M Ca** as compared that of actomyosin re-
constituted of scallop and actin. When rabbit skeletal
thin filaments were added to scallop myosin. the Mg?* -
ATPase activity was slightly inhibited in the presence
of 10°’MCa?* as compared with that of scallop thin fil-
aments. we concluded the properties of scallop thin fil-
aments were similar to that of vertebrate skeletal trop-
oninI.
1216 Physiology
Purifications and Properties of Two Isoforms of Lamprey
Skeletal Creatine Kinase. Y. Yazawa, Y. Nakajima and
J. Soga. Dept. of Nutritional Physiol., Hokkaido Univ. of
Education at Asahikawa. Asahikawa.
Two isoforms of creatine kinase, CK] and CKII, were
isolated from lamprey skeletal muscle, respectively. CK ]
was purified with ammonium sulfate fractionation, DEAE-
Toyopearl column chromatography, gel filtration, hydrox-
ylapatite column, and butyl-Toyopear! column. CKII was
Purified with ammonium sulfate fractionation, DEAE colu-
mn, gel filtration, hydroxylapatite column, and CM-Toyo-
pearl chromatography.
Two isoforms preparations appeared to be homogeneous
on SDS-PAGE and their MW of 43K was estimated from SDS-
PAGE and about 86K was estimated from gel filtration
under physiological conditions, respectively. According
to results of amino acid analysis, both isoforms conta-
ined 380 amino acids and their enzymatic properties
showed the similar results except Km values of ADP in
the reverse reaction.
EFFECTS OF ACETYLCHOLINE AND IONIC
ENVIRONMENT ON MECHANICAL PROPERTIES OF
CATCH CONNECTIVE TISSUE FROM STARFISH BODY
WALL.
T. Motokawa? and M. Nishimaru=. “Biol.
Labee shaceuot Scie lokyo sinsit won
Technol., Tokyo, “Dept. of Biol., Fac. of
Sci., Univ. of Ryukyus, Nishihara.
Connective tissue was isolated from the
body wall of the starfish Linekia
laevigata. Electron microscopical
observation revealed that the connective
tissue contained no muscle cells. Creep
tests were performed on the isolated
dermis immersed in artificial sea water
(ASW). The dermis elongated with rather a
constant rate of elongation. Creep
viscosity was defined as "stress/(strain
rate)." Acetylcholine (ACh) 10~*-10-7M
caused a biphasic response: it increased
the viscosity for a few minutes and then
the viscosity decreased to the level lower
than that before the application of ACh.
The response to ACh strongly suggested
that the mechanical properties of this
connective tissue is controlled by nerves
and thus this is catch connective tissue.
Effects of ionic environment on viscosity
also supported the suggestion. ASW with
high-K* concentration increased the
viscosity. High-Ca** ASW increased
whereas low-Ca*~ ASW decreased the
viscosity. The effects of ions were
similar to those in sea-cucumper catch
connective tissue.
COMPARISON OF CALCIUM CHANNELS IN VASCULAR
SMOOTH MUSCLES OF RATS AND FROGS.
Y.Kobayashi, K.Shinozuka and K.Hattori.
Dept. of Pharmacol., Shimane Med. Univ.,
Izumo.
Calcium channels in membrane have an
important role in intracellular signal
transduction system. In the present
study, calcium channels in vascular smooth
muscles of rats and frogs were compared.
To clarify the character of frog aorta,
catecholamines contents in vasculature were
measured by HPLC-ECD method. Adrenaline
content was significantly higher than
noradrenaline, showing apparent difference
from the predominant existence of nor-
adrenaline in mammalian vasculature.
Adrenaline-induced contraction was slightly
but significantly smaller than nor-
adrenaline-induced contraction in the same
concentration when the maximal contraction
was taken as 100 &. Adrenaline-induced
maximal contraction tended to be higher
than that by noradrenaline. Calcium
channel blockers, diltiazem and verapamil,
induced relaxation in rat isolated thoracic
aorta but not affect on adrenaline-induced
contraction of frog thoracic aorta
preparation. On the other hand, a calcium
channel blocker peptide, omega-conotoxin
relaxed frog aorta preparation, but not in
rat aorta preparation. These results
indicated that calcium channels in vascular
smooth muscles are different between rats
and frogs.
EFFECTS OF COOLING ON THE HEART BEAT OF THE
JAPANESE SPINY LOBSTER JN VIVO.
M.Nakamura, T.Kuramoto. Shimoda Marine Res.
Ctr. Tsukuba Univ., Shimoda, Shizuoka
The lobster, Panulirus japonicus, had been
acclimated in a perfusion chamber (25 x 40x
30ci, 20+1 °C) for month-periods. Electro-
cardiogram (ECG) was taken with implanted
electrodes. The Average beat rate of the
heart was about 100 bpm during spring and
summer or‘ about 50 bpm during winter. The
effect of lowered temperature on the beat
rate was studied in vivo, To cool the animal
up to 15°C, cold sea water (5°C) was poured
into the chamber. Speeds of cooling (0.1
-1.0°C /min) were dependent on volumes of the
cold water and were measured with a digital
thermometer.
In spring and summer, the beat rate de-
creased in proportion to decreases in tem-
perature by the cooling (e.g. up to 66 bpm).
However, the rate did not drop less than 60
bpm at 15 °C. In winter, the beat rate did
not follow the decrease in temperature below
18°C though it did for initial few minutes.
For example, the rate of 31 +5 bpm was held
at 18-16°C. Correlation plots between the
beat rate and the temperature obtained by 8
trials in winter showed that the beat rate
decreased for 21-19 °C and increased for 19-
16°C. The negative correlation suggests that
the lobster has some mechanisms which pre-
vent the decrease of beat rate depending on
the drop of body-temperature.
Physiology 1217
EFFECTS OF COOLING ON THE HEART BEAT OF THE
JAPANESE SPINY LOBSTER JN VITRO.
T.Kuramoto, M.Nakamura. Shimoda Marine Res.
Ctr. Tsukuba Univ., Shimoda, Shizuoka
Electrical and mechanical activities of
isolated hearts of Panulirus japonicus were
recorded simultaneously, while warm (20+ 1
°c) or cold (8-12 °C) saline was poured into
the heart via perfusion lines switched by a
faucet. The effects of lowered temperature
on the heart were studied in vitro.
Under conditions that the initial rate of
the heart beat was lower than 60 bpm, the
cooling did not always reduce the rate with
abolishing the second systolic contraction
(SSC) while increased amplitude of electro-
eardiogram (ECG). These indicated that the
effect was strong on small cardiac neurons
but little on large ones. Thus the high beat
rate under the low temperature can be pro-
duced by the large neurons, which burst at
higher rates than the small neurons.
Under conditions that the initial beat
rate was higher than 60 bpm, the cooling re-
duced both rate and amplitude of the beat
(the first systolic contraction; FSC) to 50-
40 bpm and 80-60%, respectively. Whereas,
ECG amplitude increased significantly with
cooling. Applying octopamine (a principal
pericardial hormone, 1yM) to the heart with
the cold saline, the ECG amplitude further
increased while FSC also was enhanced mark-
edly. Since the heart tension should be
feedbacked to the cardiac neurons, the en-
hanced FSC appeared to prevent the decrease
in beat rate by cooling.
THE EFFECT OF THERMAL ACCLIMATION ON THE
ELECTROCARDIOGRAM OF GOLDFISH, Carassius
auratus.
A.Morita and H.Tsukuda. Dept. of Biol.,
Fac. of Sci., Osaka City Univ.
We examined the effect of thermal
acclimation on the heart activity in vivo
by comparison of electrocardiograms (ECG)
between 10°C- and 25°C-acclimated gold-
fish. Curare and atropin were used to
exclude the influence of exercise and the
vagal regulation, respectively.
The ventricular potential difference
of 25°C-acclimated fish was significantly
lower than that of 10°C-acclimated fish
in the control, but no difference between
the two acclimation groups was found in
Curare- and atropin-treated fish.
The frequency of heart beat was sig-
nificantly higher at lower temperatures in
10°C-acclimated fish than in 25°C-
acclimated fish. This indicates tempera-
ture compensation induced during thermal
acclimation. The frequency was signifi-
cantly accelerated by injection of atro-
pin. This may be attributable to blocking
the vagal regulation by action of atropin.
The time for the auriculoventriclar
conduction of systolic impulse decreased
with temperature rising, but no Ssignifi-
cant difference was found between the two
acclimation groups. The auriculoventricu-
lar conduction rate may be independent on
acclimation temperature.
EXCITATORY AND INHIBITORY PATHWAYS FROM
MECHANICAL RECEPTORS TO CARDIO-REGULATORY
NEURONS IN BATHYNOMUS DOEDERLEINI.
K.Tanakal and K.Kuwasawa2. IDept.of Biol.,
Kyorin University School of Medicine,
Tokyo, and 2Dept. of Biol., Tokyo
Metropolitan University, Tokyo.
In Bathynomus, tactile stimuli induce
cardiac inhibition. This inhibition is
caused by reciprocal actions between two
kinds of cardio-regulatory neurons, i.e.
activation of cardio-inhibitory neurons
(CIs) and inhibition of the lst and 2nd
cardio-acceleratory neurons (CAls and
CA2s). Mechanical stimuli applied to spines
of thoracic limbs and hairs on swimmerets
caused EPSPs on CIs and IPSPs on CAs.
We have shown that activated swimmeret
movements accompanied tachycardia. In this
study, we found that the activated
swimmeret movements accompanied activation
of CAs. Extension of the thoracic limbs
and protraction of the swimmerets caused
EPSPs on CAs. There are mechanoproprio-
ceptors sensitive to retraction of the
Swimmerets. Long-lasting positive current
applied extracellularly to the receptors
caused activation of CIs and inhibition of
CAs, and long-lasting negative current
caused activation of CAs.
The results suggest that tactile
receptors are involved in cardio-inhibitory
circuits, and that mechanoproprioceptors
are involved in both the cardio-inhibitory
and -acceleratory circuits.
IDENTIFICATION OF NEURONS INNERVATING THE
CARDIOARTERIAL VALVE IN THE ISOPOD
CRUSTACEAN BATHYNOMUS DOEDERLEINI.
J.OKada and K.Kuwasawa. Dept. of Biol.,
Tokyo Metropolitan Univ., Tokyo.
The cardioarterial valves of five pairs
of lateral arteries (LA1-5) receive
inhibitory innervation from lateral
cardiac nerves (LCN1-5) originating from
3rd roots of the 4th thoracic to the 3rd
abdominal ganglia. Activation and
inhibition of LCNs induce, respectively,
dilation and constriction of the valve.
The former increases the haemolymph flow
in the LAs, and the latter decreases that.
The haemolymph distribution to LAs is
centrally controlled by combinations of
"open" and "close" modes of valve
movements among the lateral cardioarterial
valves. LCN neurons were histologically
and electrophysiologically identified in
the CNS. Back-filling of LCN5 with Cott
and Nitt revealed candidates for somata of
LCN5 neurons at the posterior half of 2nd
and 3rd abdominal ganglia. Intracellular
spikes in the soma and extracellular LCN5
impulses corresponded each other one to
one in high Mg*t saline. Injection of
Lucifer yellow into the somata showed that
the neurons actually sended axons to 3rd
roots of 2nd and 3rd abdominal ganglia,
and that they had both ascending and
descending processes in the ventral nerve
cord. It is likely that the candidates
for LCN5 neurons are cardioarterial valve
neurons themselves.
1218
GRAVITY-RECEPTION MECHANISMS OF PARAME-
CIUM
Y. Mogami, R. Tatematsu & S.A. Baba
Dep. of Biol., Ochanomizu Univ., Tokyo.
We proposed a model of gravity
reception for the negative-gravitactic
behavior of Paramecium in accordance
with the mechanism hypothesized for the
gravity-induced changes in propulsion
(Ooya et al 1992, J. exp. Biol., 163)
which includes the orientation-dependent
changes in membrane potential by the
selective activation of mechanosensory
channels differentially distributing
along the antero-posterior axis of the
cell. Computer simulation of the model,
in combination with the fact of the
potential-coupled regulation of three-
dimensional ciliary propulsive thrust
leading to the changes in the parameters
of helical swimming trajectory, demon-
strated that paramecia swim upward along
a super-helical trajectory with the axis
being parallel to the gravity vector.
Super-helical swimming paths were re-
corded from paramecia swimming under
quasi-unbound conditions in a wide
chamber. These results suggest that
Paramecium may be capable of sensing
gravity via cellular mechanoreception,
and may execute gravity-sensitive migra-
tion (gravitaxis) as well as gravity-
sensitive regulation of propulsion
(gravikinesis; Machemer et al, 1991, J.
Comp. Physiol. A, 168).
ELECTROPHY SIOLOGICAL COMPARISON OF
THERMORECEPTOR AND MECHANORECEPTOR
CURRENTS IN PARAMECIUM .
T. Tominaga and Y. Naitoh, Inst. Biol. Sci., Univ. Tsukuba,
Tsukuba, Ibaraki 305
A localized thermal stimulus applied to the anterior end of a
voltage-clamped Paramecium produced an inward
membrane current, while that applied to the posterior end
produced an outward current, when its ambient temperature
(Te) was 15°C. The reversal potentials for the anterior
current and that for the posterior current were dependent on
extracellular of Ca2+ and K+ respectively. Distribution of the
thermoreceptor mechanisms in the cell and their ion
dependence are identical with those of the mechanoreceptor
mechanisms. Primary objective of our study is to examine
whether the thermoreceptor of Paramecium share in its
mechanism with the mechanoreceptor. Effect of change in
external Nat, Lit, Mg?+, Mn2+ concentration on the reversal
potential for the thermoreceptor current was identical with
that for the mechanoreceptor current. An external application
of TEA+ reduced the posterior thermoreceptor current,
showing a dose-response relationship similar to that for the
posterior mechanoreceptor current. On the other hand Te-
dependence of the thermoreceptor current was different from
that of the mechanoreceptor current. Magnitude of the
receptor current evoked by simultaneous application of
thermal and mechanical stimuli was an algebraic sum of
magnitude of each receptor current evoked separately by each
independent stimulus. Based on these results, we discussed
the following possibilities in the relation ship between the
thermo- and mechanoreceptor systems in Paramecium 1)
The thermoreceptor channels are certainly different from the
mechanoreceptor channels, though their distributions in the
cell and ionic mechanisms are very similar with each other.
2) The thermoreceptor mechanism share the same ion
channels with the mechanoreceptor mechanisms.
Physiology
THE EFFECT OF COBALT ION ON THE
CONTROL MECHANISM OF CILIARY
ORIENTATION IN CILLIATED SHEETS FROM
PARAMECIUM.
M. Noguchi and T. Shimura. Dept. of Biol., Fac.
of Sci., Toyama Univ., Toyama.
"Ciliary reversal" induced by Ca” is cancelled
by addition of higher concentration of Co” in Triton-
glycerol-extracted Paramecium. To clarify the
action of Co”* on the control mechanism of cilary
orientation, we examined the effect of Co”* on the
ciliary response in ciliated sheets from Triton-
glycerol-extracted Paramecium. Ciliary orientation
reactivated in the presence of 1M Ca” ‘changed
from 11 o'clock to 5 o'clock by the further addition
of Co** over 2 mM (with the anterior of the cell
defined as 12 o'clock). In the presence of cyclic
nucleotides which also competed with the action
of Ca” in the ciliary response, the competing action
of Co** with Ca** became more effective. These
results indicate that Co”*expels Ca** from the
binding site and turns off the Ca”-dedpendent
controlling mechanism of ciliary beating direction.
Brief digestion by trypsin which was enough to
remove the cyclic nucleotide sensitivity did not
affect the competitive cancelation of "ciliary
reversal" by Co”. This suggests that Ca>~-
dependent regulation is essential for controlling
the beating direction, and cyclic nucleotides could
affect the mechanism secondarily.
22S DYNEIN OF PARAMECIUM MAY TAKE
PART IN THE REGULATION OF CILIARY
BEATING DIRECTION.
T. Ogawa and M. Noguchi. Dept. of Biol., Fac. of
Sci., Toyama Univ., Toyama.
Ciliary response to either Ca” or cAMP was lost
by a digestion with high concentration trypsin in
the Triton-glycerol-extracted Paramecium. To
determine which component of axonemal
polypeptides is essential for Ca** and cAMP
dependent controlling mechanism of cilia, we
examined the change in SDS-PAGE pattern and
ATPase activity of the axonemes during the course
of thé trypsin digestion. A band of the highest
molecular weight polypeptieds in SDS-PAGE
pattern of axonemes disappeared by the trypsin
digestion within 20 seconds. ATPase activity of
ciliary axonemes also decreased and reached a
minimum level (approximately 50% of activity of
non-digested axonemes) within 20 seconds.
Trypsin-treated axonemes were extracted with 0.6
M KCI and the extract was separated with sucrose
density gradient centrifugation and fractionated.
SDS-PAGE pattern of each fraction indicated that
the high-molecular weight peptide was one of the
heavy chain of 22S dynein. This result suggests
that 22S dynein plays an important role in the
regulation of ciliary beating direction.
Physiology 1219
EFFECTS OF METHANOL ON THE
INHIBITORY ACTION OF VANADATE AND
GLYCEROL ON AXONEMAL ATPase.
T. Atago and M. Noguchi. Dept. of Biol., Fac. of
Sci., Toyama Univ., Toyama.
Axonemal ATPase from eukaryotic flagella and
cilia is inhibited by vanadate and glycerol. On the
contrary, methanol induce unphysiological
enhancement of the ATPase activity which
accompanies inability of sliding between outer
doublet microtubules. To examine the relation
between the stimulating effect of methanol and
the inhibitory effects of vanadate and glycerol, the
ATPase activity of axonemes from Chlamydomonas
flagella was measured in the presence of methanol
in addition to vanadate or glycerol.
Methanol which is supposed to accelerate
product release from dynein:ADP-Pi activated the
axonemal ATPase in some extent in spite of the
presence of the inhibitors. The ratio of
dynein:ADP-Pi to dynein:ADP-Vi inferred from the
assumption that ATPase activity is proportional
to the concentration of dynein-ADP-Pi complex
increased with increasing methanol concentration.
This might suggest that dynein-ADP-Vi state
become unstable as well as dynein-ADP-Pi state.
DISCRETE NATURE OF FLAGELLAR BENDING
S. A. Baba, M Imagawa and Y. Mogami.
ep. of Biol., Ochanomizu Univ., Tokyo.
Present address: Dep. of Mol. Biol., Nagoya
University, Nagoya
Bending shapes of live and demembranated
sea-urchin and starfish sperm flagella were
studied at high resolution by means of digital
image analysis. As reported previously (Baba,
S. A., Mogami, Y. & Nonaka, K. 1990. In Biologi-
cal Motion (Lecture Notes in Biomathematics 89)
ed. M Alt & G Hoffmann, Springer-Verlag, Ber-
lin, pp. 145-154; Baba, S. A., Hoshino, Y. &
Mogami, Y. 1991. In Comparative Spermatology 20
Years After, ed. B. Baccetti, Raven Press, New
York, pp. 333-336), the curvature of a flagellum
tended to change abruptly from one value to
another along the flagellum, whereas it remained
constant within a segment of variable length.
The length of the segment of this quasi-stable
bend form, in which the curvature is kept con-
stant transiently, varied in a beat cycle and
among different flagella of the same species,
but the value of that curvature itself appeared
to be invariant in flagella at least within the
same species. Both the maximum and the average
curvature of either the principal or the re-
verse bend of sperm attached to the cut edge of
agar gel by the head were larger than those of
sperm swimming freely, while quasi-stable bend
forms appeared of common curvatures, suggesting
that discrete bending described here reflects
an inherent structural feature of flagella
MECHANISM OF CALCIUM-INDUCED REVERSAL OF
DIRECTION OF ROTATIONAL MOVEMENT IN REAC-
TIVATED SEA URCHIN SPERMATOZOA.
S. Ishijima and Y. Hamaguchi, Biological
Laboratory, Faculty of Science, Tokyo
Institute of Technology, O-okayama, Megu-
ro-ku, Tokyo.
Examination of demembranated sea urchin
sperm has shown that Ca * in a reactiva-
tion solution regulates the direction of
yaw (rotation of a sperm about the axis
perpendicular to the beat plane); a clock-
wise (CW) yaw under the coverslip dominat-
ed in high Ca *+ concentrations and an
anticlockwise (ACW) yaw in low Ca con-
centrations. There is a one-to-one corre-
spondence of direction between yaw and
roll (rotation of a sperm about its long
axis); a CW yaw under the coverslip corre-
sponded to a CW roll as viewed from the
anterior end of sperm and an ACW yaw
corresponded to a ACW roll. _Therefore, a
CW roll dominates in high Ca = goncentra-
tions and an ACW roll in low Ca*t concen-
trations. Since the direction of roll is
determined by the sense of the three-
dimensional components of bending waves,
sperm beat with right-handed bending waves
in high Ca * concentrations and they beat
with left-handed ones in low Ca¢%* concen-
trations. This calcium-induced conforma-—
tional change in the axoneme is perhaps
caused by a reversal of the direction of
propagation of the local active sliding
between doublets around the axoneme.
EFFECT OF ATP CONCENTRATION ON THE
VELOCITY OF MICROTUBULE SLIDING IN
REACTIVATED SEA URCHIN SPERM FLAGELLA
UNDER IMPOSED HEAD VIBRATION.
C. Shingyoji!, K. Yoshimural, D. Eshel2, I. R.
Gibbons”, and K. Takahashi3. 1Zool. Inst., Fac. of
Sci., Univ. of Tokyo, Tokyo, 2Pacific Biomedical
Research Center, Univ. of Hawaii, U.S.A., and
3Dept. of Biology, Int. Christian Univ., Tokyo.
The flagellum of a sea urchin sperm whose head
is held with a suction pipette and vibrated laterally
beats in synchrony with the imposed vibration.
Similar synchronized stable beating was obtained in
sperm demembranated and reactivated with 10 uM-4
mM ATP. We analyzed the sliding velocity of
reactivated Tripneustes gratilla sperm under
various vibration frequencies. At ATP
concentrations lower than 100 1M, the apparent
time-averaged sliding velocity of axonemal
microtubules, obtained as twice the product of
frequency and bend angle, decreased with the beat
frequency. At ATP concentrations above 100 uM , the
sliding velocity decreased with the beat frequency
only below the beat frequency of the unvibrated
flagella. When the beat frequency was equal to or
higher than the beat frequency of the unvibrated
flagella at the same ATP concentration, the sliding
velocity remained unchanged. These results suggest
that the sliding velocity of axonemal microtubules
does not depend solely upon the local concentration
of ATP, but is also controlled by an oscillatory
mechanism closely associated with the mechanisms
for the initiation of bending waves.
1220 Physiology
FORCE-VELOCITY RELATION OF THE ATP-DEPENDENT
KINESIN-MICROTUBULE SLIDING.
H. Sugi!, I. Takagi!, K. Oiwa! and T. Shimizu2.
1Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo and
2Biol. Eng. Dept., Res. Inst. of Polymers and Textiles,
Tsukuba.
When the tip of a glass microneedle, whose tip
is coated with kinesin, is made in contact with a
single demembranated axoneme isolated from sea-
urchin sperm in the presence of ATP, the needle
moves along the axoneme in one direction. If the
needle is at right angles to the axoneme long axis, it
moves for several microns in 10-20 s at room
temperature (23-25°C) until it eventually stops
moving. Judging from the elastic coefficient of the
needle, the maximum force generated by kinesin-
microtubule sliding is several pN.
Assuming that the needle stop moving on the
axoneme when the maximum "isometric" force
generated by knesin-microtubule interaction
balances with the force exerted by the bent needle,
we tentatively obtained the force-velocity relation of
the ATP-dependent kinesin-microtubule sliding by
recording the needle movement with a video system.
The force-velocity curve thus obtained is nearly
straight and differs from that of the ATP-dependent
actin-myosin sliding obtained under similar
auxotonic conditions.
TWO-STEP ACTIVATION OF MOUSE SPERM MOTILITY
Y.Si and M. Okuno, Dept. of Biol., Coll. of
Arts and Sci., Univ. of Tokyo, Tokyo.
To examine the activation of mouse sperm
motility, the state of flagellar axoneme in
the intact sperm was further investigated
using model sperm. When dry sperm from
cauda epididymis was diluted into sucrose
solution, the sperm showed little motility
with the velocity of 13.5+3.8 ywm/sec and
beat frequency of 1.5+0.4 Hz. Then, the
sperm was transferred to the extraction
solution for demembranation. The demembra-
nated sperm was immotile in the absence of
cAMP. We referred to this kind of intact
sperm as "Initiated" one. On the other
hand, when dry sperm was diluted into
either 155 mM NaCl solution or sucrose
solution containing 2 mM Ca“*, the sperm
showed the beat frequency of about 9 Hz.
The demembranated sperm was motile in the
absence of cAMP. We referred to this kind
of intact sperm as "quasi-activated" one.
Alternatively, when dry sperm was diluted
into the sucrose solution containing 20 mM
HCO, , the sperm exhibited a vigorous
motility with the velocity of 171.2+10.1
um/sec and beat frequency of 11.3+1.2 Hz.
The demembranated sperm was also motile in
the absence of cAMP. We referred to this
kind of intact sperm motility as "Activat-
ed" one. Our results indicated that the
activation of mouse sperm motility took a
two-step process and the conversion of
quasi-activated motility to activated
motility was highly dependent on the
presence of bicarbonate.
EFFECTS OF CALCIUM AND CYCLIC AMP ON YAWING
DIRECTION OF HAMSTER SPERMATOZOA.
K.Ishida’, T. Umeda and M. Okuno“~. SBept.
of Urol., Teikyo Univ. Sch. Med. and*“Coll.
of Arts and Sci., Univ. of Tokyo, Tokyo.
Yawing direction can be easily detect-
ed in hamster spermatozoa since the shape
of the acrosome is asymmetry. We defined
the yawing direction "right" to be the
follwing hook direction of the acrosome for
convenience. When the extracellular calcium
concentration was below 10-4, the yawing
direction of the cauda epididymal spermato-
zoa of the hamster was permanentry "left"
after dilution. On the contrary, the direc-
tion was "right" upon dilution when the ex-
Eracelluyar calcium concentration was higher
than 10 *M. However, the direction gradually
turned to "left" and that of every spermato-
zoa became “left" finally. The direction
could never be returned from "left" to
"right" again by further elevation in cal-
cium concentration after the spermatozoa
once changed to the direction from “right”
to "left". The higher the extracellular cal-
cium concentration raised, the longer the
direction was continued to be "right". The
yawing direction was permanentry “right"
when the membrane permeablecyclic AMP deri-
vative, 8-bromo cyclic AMP, was present
above 1mM even in calcium-free dilution
medium. As well as the effect of the 8-bromo
cyclic AMP, the yawing direction never
changed from "right" to "left" when theophy-
lline was added above 0.75mM in calcium-,
and 8-bromo cyclic AMP-free dilution medium.
INITIATION OF SPERM MOTILITY IN RELATION TO
ACTIVITY OF MITOCHONDRIA.
M. Okuno & Y. Si. Dept. Biol., Coll. Arts &
Sci., Univ. Tokyo, Tokyo.
Initiation of sperm motility has been
considered to involve a cAMP dependent
process such as phosphorylation of 15K
protein in salmonid fishes. However, the
role of the phosphoproteins in the axoneme
was unclear. We looked at the mitochondrial
activity using a fluorescent dye, DASPEI,
which stains active mitochondria.
Salmonid spermatozoa were immotile in the
presence of KCl and appeared very low
mitochondrial activity. When they were
diluted in K'-free medium the mitochondria
displayed strong fluorescence. Mitochondria
were,also active in oyster and barnacle when
spermatozoa were diluted in sea water and
initiated motility. Hamster spermatozoa
shows low activity in the absence of Ca’'
(about 0.8 Hz in flagellar beat frequency).
In this’ state, the mitochondria were
relatively active. In the presence of high
concentration of Ca!’ flagella were activated
(about 10 Hz) and the mitochondria were also
active. On the other hand, when spermatozoa
were incubated with CCCP or other inhibitors
of mitochondria in the presence of
theophylline flagella kept intermediate
motility (3 Hz) although the mitochondria
were inactive. These results suggested that
sperm flagellar activation involved at least
two factors, one was a cAMP dependent
conversion of axonemal motile system and the
other the activation of mitochondria for
supplement of ATP to the motile system.
Physiology 1221
INITIATION OF SPERM MOTILITY IN MARINE TELEOST:
ROLES OF INTRACELLULAR K* AND CA” .
H. Takai and M. Morisawa
Misaki Marine Biological Station, Fac. of Sci., Univ. of Tokyo.
Kanagawa. :
Spermatozoa of marine teleosts are immotile in the male
Teproductive organ by isotonic osmolality of the seminal plasma,
and that motility initiation occurs when they are spawned into
hypertonic seawater (Morisawa and Suzuki 1980). Recently, it
has been reported that intracellular pH(pH,) and intracellular
Ca**(Ca**,) increase during the motility initiation in hypertonic
solution in puffer and flounder sperm (Oda and Monsawa 1990).
In addition, we found here that plasma membrane of puffer
sperm was depolarized at the time of the motility initiation.
From these facts, it seems possible that increases in pH, and
Ca. and membrane depolarization have some roles in the
initiation of sperm motility. However, it is still unclear whether
pH, and membrane potential directly participate in the motility
initiation process because similar membrane depolanzation and
increase in pH, were observed in the immotile sperm in the
presence of monensin or nigericin in isotonic NaCl solution. In
addition, initiation of sperm motility did not occur when mem-
brane depolarization was caused by gramicidin. Futhremore, it
was found that puffer sperm which were quiescent in isotonic
KCI solution, became motile by the addition of K* ionophore,
nigericin. In contrast, spermatozoa were still immotile in isotonic
NaCl even if nigericin was supplemented. Monensin had no
effect on the sperm motility in isotonic solution. These results
suggest that increase in intracellular K* is a candidate for the
trigger for the initiation of sperm motility. It is possible that
increase in intracellular K* and Ca” participate in the initiation
of sperm motility in marine teleosts.
CONTROL OF CHEMOTAXIS IN AMOEBA CELLS OF
THE CELLULAR SLIME MOLD, Dictyostelium
discoideum I. THE ASSAY SYSTEM
T. Tani and Y. Naitoh Institute of Biological
Sciences, University of Tsukuba, Tsukuba 305
Amoebae of cellular slime mold Dictyostelium have
been known to exhibit positive chemotaxis to cAMP.
We developed a novel assay system for quantitative
examination of chemotaxis in the amoebae. The
assay system consists of three thin (0.17 mm)
compartments, 1) for cAMP-containing solution
(18x18 mm), 2) for the reference solution (18x18
mm), and 3) for amoeba-containing solution (0.4x18
mm). These three compartments are put together
placing the amoeba-containing compartment
between two other compartments. Thus amoebae in
the central compartment are subjected to a solution
with a definite concentration gradient. Amoebae
were tape-recorded and time course of change in
their cell shape, moving direction, moving velocity
and of their distribution in each compartment were
examined on replayed images. The amoebae showed
a transient stoppage of their locomotion due to
retracting of their pseudopodia upon subjection to a
solution with a concentration gradient of cAMP.
Then they resumed their locomotion towards test
solution with cAMP. Velocity of the locomotion was
higher in a test solution with higher cAMP
concentration by 108 M, while it became lower
when cAMP concentration was as high as 10°© .
Involvement of the orientation response as well as
the locomotor velocity in establishment of
accumulation of the amoebae are discussed.
CONTROL OF ALL-OR-NOTHING TYPE CONTRACTION IN THE
STALK BY CALCIUM AND RELATED SUBSTANCES IN
VORTICELLA.
K. Katoh and Y. Naitoh. Inst. of Biol. Sci., Univ. of
Tsukuba, Ibaraki
a
A peritrich ciliate, Vorticella exhibits an all-or-
nothing type contraction, involving coiling of its stalk
and shrinkage of its cell body, in response to a stimulus.
Coiling of the stalk always starts from the portion near
the cell body and propagates down the stalk. Contractile
element of the stalk is known to be activated by Ca?+.
An injection of Ca*+ buffer-containing solution into
the cell body caused coiling of the stalk to quasi-
maximum extent, when [Ca*+] in the buffer was higher
than 5x10-°M. Simillar injection caused no coiling when
the [Ca?+] was lower than 5x10-8 M. This indicates that
coiling of the stalk was induced when [Ca?+] in the cell
body was over a certain threshold value (5x10-® M).
5x10-8 M Ca?* did not acivate contractile element of the
stalk in triton extracted Voticella. These results suggest
that Ca?* injected into the cell body induced Ca?+ release
from some Ca?* storage site to evoke coiling in the stalk.
An endoplasmic reticulum containing Ca?+ has been
reported to be present along the contractile element in
the stalk. We found that an injection of caffeine- (50
mM) and ryanodinc- (0.2-2 4M) containing solution into
the cell body caused all-or-nothing type contraction,
that an injection of procaine- (5 mM) and ruthenium
red- (10 4M) containing solution into the cell body
inhibited contraction and that an injection of IP,- (100
uM) containing solution into the cell body was not
effective for evoking contraction. The "Ca?+-induced Ca?+
teleasc" might be responsible for all-or-nothing
property of the stalk contraction in Vorticella.
CHOLINERGIC MECHANISMS IN THE HEART OF THE
CHITON (LIOLOPHURA JAPONICA).
S. Matsumura and K. Kuwasawa. Dept. of
Biol., Tokyo Metropolitan Univ., Tokyo.
Using the chiton in the lowest
molluscan taxon, we investigated effects
of acetylcholine (ACh) on the heart and
cholinergic blockers on the ACh effects.
ACh (>107M) exerted negative chronotropic
and inotropic effects on the heart. The
membrane potential of the auricular
myocardium during ACh-induced cardiac
arrest was almost the same as the maximum
diastolic hyperpolarization of a cardiac
cycle. The membrane potential of
ventricular muscle cells during the ACh-
induced cardiac arrest was always more
hyperpolarized than the maximum diastolic
membrane potential of the cells. Cardio-
inhibitory responses induced by
stimulation of central nerve cords were
blocked by methylxylocholine (MX), and
blocked by tubocurarine (TC) only in the
auricle. TC antagonized inhibitory ACh
effects on the auricle, but not
antagonized ACh effects on the ventricle.
MX antagonized inhibitory ACh effects on
both the auricle and ventricle. It is
likely that the ACh-induced cardiac
inhibition is mediated by TC-sensitive and
MX-sensitive ACh receptors in the auricle,
and by MX-sensitive ACh receptors in the
ventricle. We may conclude that
cholinergic neural inhibitory control of
the heart known in other molluscs is
extended to the taxon at the phylogenetic
beginning of the Mollusca.
1222 Physiology
EFFECTS OF BIOGENIC AMINES ON THE ADULT
HEART OF THE ISOPOD CRUSTACEAN LIGIA
EXOTICA
H. Yamagishi and Y.Terano, Inst. of Biol.
Sci., Univ. of Tsukuba, Tsukuba
ee
Effects of biogenic amines (serotonin,
dopamine, octopamine) on the heart beat
were examined in the neurogenic heart of
adult Ligia. All the three amines could
couse the frequency of burst discharge to
increase in the cardiac ganglion and
accerelate the neurogenic heart beat. The
effect was less potent by dopamine than by
the others. The frequency increase was
concentration dependent and the threshold
ranged from 10-9 (cerotonin, octopamine) to
10-8 M (dopamine). Intracellular recodings
confiemed the accerelatory effects of all
the amines on _ the spontaneous burst
activity of the cardiac ganglion cells.
Dopamine could also cause the magnitude
of contraction to increase in the heart
beat. With application of dopamine the
heart beat first increased in frequency and
then increased gradually in beat amplitude.
The increase in beat amplitude lasted
longer after changing perfusate to the
normal saline. Intracellalar records from
the heart muscle showed that the
membrane depolarization underlying the
potential respones’ to the ganglionic
discharges gradually increased by
application of dopamine. These results
suggest that dopamine affects not only the
cardiac ganglion but also the heart muscle
EFFECTS OF CRUSTACEAN PEPTIDE HORMONES ON
THE CARDIAC GANGLION AND MYOCARDIAL CELLS
IN THE HEART OF HERMIT CRAB, ANICULUS
ANICULUS.
T.Yazawa,K.Tanaka* and K.Kuwasawa. Dept.of
Biol.,Tokyo Metropolitan Univ.and *Dept.of
Biol., Kyorin Univ. School of Med., Tokyo.
Proctolin, crustacean cardio-active
peptide, red pigment concentrating
hormone, adipokinetic hormone, and
FMRFamide were administered to cut-opened
heart preparations. Electrical activities
of small and large ganglionic neurons and
myocardial cells, and contraction force of
the heart were simultaneously recorded
while the preparations were perfused with
the peptides. Potency of proctolin was
103-104 times greater than that of other
peptides. Proctolin (10-12 M - 10-& M)
showed inotropic effects, but little
changed heart rate, i.e. ganglionic burst
rate. Intra-burst impulses of both small
and large ganglionic neurons were
increased. Positive tonotropic effects
were elicited by proctolin at higher
concentrations (>10-8 M). Membrane
responses were superimposed on compound
EJPs by proctolin application (10-9 M).
However, current-induced electrical
responses, which were blocked by either
TTX or Ca?+-free salines, were not
enhanced by proctolin (10-8 M). Membrane
resistance of the myocardium was not
changed by proctolin (10-® M). It is
likely that ganglionic excitation was the
most important action of proctolin in its
excitatory effects on the heart.
MECHANISM OF CHANGES OF ANSERINE AND
CARNOSINE LEVEL AND THEIR RATIO IN RAT
FAST AND SLOW TWITCH MUSCLES.
M.Tanaka.Dept.of Physiol.,St.Marianna
Univ.Sch.Med. ,Kawasaki.
"Changes in carnosine and anserine
levels and their ratio during development
and the effect of endurance training on
these peptides levels were studied in rat
fast and slow twitch muscles. It has been
reported that in the leg muscles of the
chronic infected rats,the carnosine level
decreases without any change in the
anserine level,while tissue free histidine
level increases 10-fold. The endurance
training decreased both the peptides
levels especially the anserine level
(Carnosine; p < 0.01, Anserine; P < 0.001)
,but had no effect on the histidine nor
other amino acids examined in both fast
and slow twitch muscles. There are two
possibilities of the decrease of both
peptides levels by endurance training.
One is the decrease of carnosine
synthesis and the other is an increase of
its degradation.
REGULATION OF BUCCAL MUSCLE CONTRACTION BY
A PEPTIDE-CONTAINING MOTONEURON IN THE
SNAIL ACHATINA FULICA
M. Yoshida and M. Kobayashi. Physiol.
Lab., Fac. of Integrated Arts and Sci.,
Hiroshima University, Hiroshima.
In the buccal ganglia of the African
giant snail, we identified a pair of exci-
tatory motoneurons of the radula protractor
and named them B10s. Muscle fibers of the
protractor responded to B10 firing with
unitary excitatory junction pontentials
(EJPs). Bl0-evoked EJPs and contractions
of the protractor were blocked by applica-
tion of a cholinergic blocker propantheline
to the muscle. Contractions of the
protractor induced by direct application of
acetylcholine (ACh) to the muscle were also
blocked by propantheline. Thus the main
excitatory transmitter of B10 may be ACh.
Immunohistochemical examination revealed
that the substance like ACEP-1 (Achatina
cardio excitatory peptide-1l), which is a
bioactive peptide isolated from Achatina,
was located in Bl0s. The nerve terminals
in the protractor also showed ACEP-1-like
immunoreactivity. Application of ACEP-1 to
the protractor enhanced Bl0-evoked EJPs and
contractions of the muscle, whereas ACh-
induced contractions of the muscle were not
affected by the peptide. These observa-
tions suggest that ACEP-1 is released
together with ACh from B10 in certain con-
ditions and acts on prejunctional terminals
of B10 to increase the release of ACh.
Physiology 1223
LOCALIZATION OF NEUROPEPTIDES ISOLATED FROM
A PULMONATE SNAIL, ACHATINA FULICA.
M.Fujiwara-Sakata and M.Kobayashi
Physiol. Lab., Fac. of Integrated Arts and
Sci., Hiroshima Univ., Hiroshima.
Several neuropeptides concerned with the
regulation of heart beat have been isolated
from the central nervous system (CNS) and
the heart of Achatina fulica. They include
ACEP-1(SGQSWRPQGRFa) and FMRFa. The
localization of these peptides in CNS and
the heart: was examined immunohistochemical-
ly. In the right cerebral ganglion, more
than 50 neurons about 30 1m in diameter
showed ACEP-1-like immunoreactivity, and
many of them also showed FMRFa-like
reactivity. Axons from these cells mainly
went down to the right cerebro-pleural and
cerebro-pedal connectives. In the surface
area of the subesophageal ganglia, a number
of small neurons showed FMRFamide-like
immunoreactivity. In deeper layers of the
ganglia, several identifiable neurons
showed FMRFamide- or ACEP-1-like
reactivity. The atrium and aortic region
of the ventricle had both FMRFamide- and
ACEP-1-like materials. Whole mount
preparation of the heart demonstrated that
ACEP-1-immunoreactive nerve fibers ran to
the atria abundantly, and a few small
fibers entered into the ventricular wall.
It seems that neuropeptides in nerve fibers
in the atrium are released through small
nerve fibers to the ventricle, where they
exhibit their modulatory action.
SEROTONERGIC PRESYNAPTIC INHIBITION IN THE
GILL OF APLYSIA SPECIES.
M. Kurokawa and K. KuwaSawa. Dept. of
Biol., Tokyo Metropolitan Univ., Tokyo.
As, in gills of Aplysia kurodai and
A. juliana, we have shown that terminals
of motor axons running from the abdominal
ganglion to the efferent branchial vessel
received presynaptic inhibition, we tried
to specify the transmitter responsible for
the presynaptic inhibition in this study.
Fine processes immunoreactive to anti-
serotonin antiserum were found to branch
out from the branchial nerve and to
innervate musculatures of the efferent
branchial vessel. Serotonin was found to
depress twitch contractions of the vessel
evoked by stimulation of the branchial
nerve when the branchial inner cavity was
perfused with a solution containing the
drug. Excitatory junctional potentials
(EJPs) recorded from muscle cells of the
vessel induced by motor axons arising from
the abdominal ganglion were also depressed
by serotonin. Methysergide antagonized
the serotonergic depression of both twitch
contractions and the EJPs in the vessel.
The presynaptic inhibition was also
antagonized by methysergide. The results
may show that the presynaptic inhibition
is mediated by serotonergic axons running
from the abdominal ganglion.
STRUCTURE AND ACTION OF A MYOMODULIN-CARP
FAMILY PEPTIDE ISOLATED FROM AN ANNELID,
PERINEREIS VANCAURICA.
T.Takahashil, Y.Muneokal, O.Matsushima2, I.
Kubota?, T.Ikeda4, H.Minakata4, K.Nomoto4,
T.Nose> and W.Miki2. lrac. Integrated Arts
and Sci., Hiroshima Univ., Hiroshima,“Zool.
Inst., Fac.Sci., Hiroshima Univ., Higashi-
Hiroshima, 3Suntory Bio-Pharma Tech Center,
Gunma, 4suntory Inst. Bioorganic Res. ,Osaka
and ~Marine Biotech. Inst., Shimizu.
Myomodulin-=CARP family peptides are
widely distributed in molluscs. In the
present study, we found a member of the
family in the annelid Perinerets vaneaurica
The structure of the peptide is AMGMLRM-—
amide. That is, the peptide has M-amide at
its C-terminus, though all the molluscan
peptides have L-amide. On phasic contraction
of the ABRM of Mytilus by repetitive
electrical stimulation,the annelid peptide,
as well as the molluscan peptides, showed a
potentiating effect at lower doses and an
inhibitory effect at higher doses. On catch
tension of the ABRM, the annelid peptide,
as well as the molluscan peptides, showed a
marked relaxing effect. For these effects,
substitution of the C-terminal L-amide of
the molluscan peptides with an M-amide
seemed not to be deliterious. On the
esophagus of Peritnerets,all of the family
peptides showed a contractile effect. The
annelid peptide was the most potent . Sub-
stitution of the L-amide of the molluscan
peptides with an M-amide seemed to increase
the contractile potency.
THREE MIP (MYTILUS INHIBITORY PEPTIDE) -
RELATED PEPTIDES ISOLATED FROM THE VENTRAL
NERVE CORDS OF AN ECHIUROID WARM, URECHIS
UNICINCTUS.
T.Ikedal, K.Nomoto!, I.Kubota2, W.Miki3, Y.
Muneoka*. lsuntory Inst. Bioorganic
Research, Osaka Suntory Bio-Pharma Tech
Center, Gunma, auamine Biotechnology Inst.
Shizuoka and 4Fac. Integrated Arts and Sci.
Hiroshima Univ., Hiroshima.
More than 25 species of MIPs have been
isolated from the molluscs, Mytilus, Helix
and Achatina. All MIPs show a potent
inhibitory effect on contractions of the
anterior byssus retractor muscle (ABRM) of
Mytilus. From the study of structure-
activity relationships of MIPs, the essen-—
tial part for the activity is FVamide, and
proline residue is also important. In
fact, all MIPs have the —PXFV(I)amide
structure in their C-terminal portions as a
common sequence. Recently a MIP-related
peptide, MRYFVamide, which lacks the pro-
line residue, was isolated from Mytilus
(Fujisawa unpublished).
In the present study, we purified three
peptides which are closely related to
MRYFVamide from the ventral nerve cords of
an echiuroid warm, Urechis unicinctus.
Their structures were determined to be as
follows: Ala-Arg-Tyr-—Phe-Leu-NH9
Ala-Lys-Tyr-—Phe-Leu-NH9
Ala-Lys-—Phe-Phe-Leu-NH9
These peptides show inhibitory effects on
contractions of inner circular body-wall
muscle of Urechis and ABRM of Mytilus.
1224 Physiology
EXTENSION EVOKING DESCENDING INTER-
NEURONS MODIFY THE UROPOD MOTONEURONS
ACTIVITY
H. Namba, T. Nagayama and M. Hisada
Zool. Inst., Fac. of Sci., Hokkaido Univ., Sapporo.
Uropod motor pattern of the crayfish avoidance
reaction is modified during the fictive abdominal
extension. We have determined the abdominal
extension evoking interneurons are responsible to the
modification of the uropod motor neuron activity via
premotor nonspiking interneurons.
Among 28 extension evoking interneurons (EEIs) so
far could be picked up at the superficial lateral bundle
of abdominal connective, 10 EEIs yielded enough data to
characterize them according to their effects on the
abdominal extension motoneurons and the uropod
motoneurons. Six of them can be classified into a group
of EEI with effects both on slow extensor motoneurons
and uropod motoneurons. The rest (4) showed effected
to the extensor motoneurons but none to the uropod
motoneurons. EEI with the effects on the slow extensor
motoneurons and uropod motoneurons inhibited closer
and/or excited opener motoneurons. Morphologically,
these EEIs projected into the dorsal region of the
neuropile in the terminal abdominal ganglion. Their
dendritic fields overlapped with the nonspiking
interneurons but not with the sensory neurons which
projected into the ventral region of the neuropile. EEIs,
thus, are likely to modify the nonspiking interneurons
response to the sensory stimulation of the tailfans
directly but not the sensory terminals. Possibility of
EEIs having also the parallel direct connection to the
uropod motoneurons remains to be tested.
OUTPUT EFFECTS OF ASCENDING INTERNEURONS
UPON THE ABDOMINAL POSTURAL SYSTEM IN
CRAYFISH
H. Aonuma, T. Nagayama and M. Hisada
Zool. Inst., Fac. of Sci., Hokkaido Univ., Sapporo
Majority of ascending interneurons that originate
from the terminal ganglion receives sensory
imformations from the tail fan and they have output
effects upon closer and openner motoneurons in the
ganglion. About 30 ascending interneurons have so far
been identified by the physiological and morphological
characteristics in the terminal ganglion. These
ascending interneurons project their axons to anterior
ganglia, Output effects upon abdominal postural system
and the structures of branches in the anterior
abdominal ganglia have been studied.
Intracellular recordings were made _ using
microelectorodes filled with 3 % neurobiotin.
Extracellular signals were simultaneously recorded
using pin electrodes. They were placed on all extensor
and flexor motoneurons of each abdominal ganglion
and closer and openner motoneurons of the terminal
ganglion.
This simultaneous multirecording study revealed that
majority of ascending interneurons had output effects
upon extensor and flexor motoneurons. These effects
are the same in all of abdominal ganglia. Neurobiotin
Staining revealed that ascending axons spreaded their
branches in each abdominal ganglion. These branches
innervated ipsilateral neuropil of each only.
These results indicated that the majority of ascending
interneurons cooperatively controlled extensor and
flexor motoneurons of all abdominal ganglia and that
contralateral cooperation relied on _ indirect
interactions in each ganglion.
ACETYLCHOLINE RECEPTOR CHARACTERISTICS IN A
SENSORY NONSPIKING INTERNEURON OF CRAYFISH.
M. Takahata, H. Nakamura and M. Niwa. Sect. Neuro-cybem.,
Res. Inst. Elect. Sci., Hokkaido Univ., Sapporo.
nal abdominal ganglion of the crayfish Procwnbarus clarkii
Girard, was affected by perfusion of acetylcholine (ACh)
agonists and antagonists using the isolated abdominal nervous
system preparation. The synaptic response of LDS to electrical
stimulation of the third root sensory bundle was recorded by a
Lucifer-filled glass microelectrode penetrating the cell on the
midline. Identification of the cell was based on its morphology
revealed by Lucifer yellow injection.
The synaptic delay between the sensory neurons and LDS
was less than Imsec, suggesting their monosynaptic connection.
The half decay time of LDS response was about 10msec at the
resting potential (N=46; -70+10mV). By perfusing the
preparation with Ca-free, Mg-rich (5 to 10 fold) saline, the
synaptic response of LDS disappeared completely and restored
later in the normal saline. The response was reduced by artificial
depolarization and enhanced by hyperpolanization with the
reversal potential at about -16mV. These results indicate that the
LDS response is mediated chemically. Perfusion of 10mM ACh
under Ca-free, Mg-rich condition caused depolarization (10-
20mV) of LDS and reduction of its synaptic response. 0.1mM
nicotine showed similar effects but 1mM muscarine had no
visible effect. The LDS response was blocked by 1mM D-
tubocurarine and 20mM hexamethonium. However, 1mM
atropine was effective as well. The results suggest that the
dendritic membrane of LDS has ACh receptors which resemble
the ionotropic nicotinic receptor of vertebrates but differ in
atropine sensitivity.
THE MECHANISM OF RHEOTAXIS IN THE WATER
STRIDER, GERRIS PULUDUM.
K. Taneda, M. Sakagami and T. Matsuoka. Dept. of
Biol. Fac. Sci. Kochi Univ., Kochi
The rheotactic behavior of the water strider,
Gerris puludum was recorded on VTR. The record
was played and _ projected on a _ digitizer
board (Graphtec, KD3300) through a video pro-
jector (Fujix, HP-40Hi). Six points of the body
for analyzing the position of body center and
bending angles of legs were entered using a
reposed picture of the record. The digitized data
were analyzed and were represented graphically.
Time-changes in the bending angle of each leg and
in the position of body center were measured
using a blinded, antennae- or cerci- amputated
specimen. Each specimen as well as untreated one
showed conspicuous rheotactic behavior. There-
fore, compound eyes, antennae and cerci seem to
play no role in the behavior. When the specimen
directed upstream, the middle legs on both sides
always showed synchronized movements. While
when the specimen directed downstream or at a
right angle to the stream, it moved its one side
of the middle leg and turned to upstream. Thus,
the middle legs seem to play an important role in
upstream orientation.
Physiology 1225
IDENTIFICATION OF SENSORY NEURONS
RESPONSIBLE FOR THANATOSIS IN THE
CRICKET Gryllus bimaculatus.
H. Nishino and M. Sakai, Dept. of Biol., Fac. of Sci.,
Okayama Univ.,Okayama.
The cricket Gryllus bimaculatus shows a
maintained flexion posture in response to a
pressure on the prothorax (Zool. Sci. 1987, 1991).
We searched the prothorax for sensory neurons
receiving pressure input and triggering
thanatosis. Transection of the nerve 2 (N2)
emanating from the antero-ventral part of the
prothoracic ganglion abolished thanatosis but that
of the other nerves did not. The N2 branched off
into 8 thinner bundles which innervated cuticles
and apophysis on the ventral side of the neck and
thorax. A total of 14 somata were found on 3
bundles near the branching point. They had short
dendrites and genta projecting axons which
extended to the aratnaracies suboesophageal,
eeeatioracic. and metathoracic ganglia. No
efferent axons were present in the N2.
Mechanical stimulation of the isolated N2
including 14 neurons in question evoked both
phasic and tonic spike responses. These stretch
receptor-like neurons may be responsible for
triggering and maintaining thanatosis.
THE MUSHROOM BODY OF INSECT BRAIN PARTICI-
PATES IN SPATIAL MEMORY PROCESSING.
M.Migunami J.M.Weibrecht N.J.Straus-
feld“. lpept. Bil@il, 5 Wee>s Sile, kanrnginiy
Univ., Fukuoka and “Arizona Research Labs,
Div. of Neurobiol., Univ. Arizona, Tucson.
Many insects, including bees and ants,
possess a well-developed spatial memory.
We report that the mushroom bodies (MBs)
of cockroach brain participate in the
processing of spatial memory.
Cockroaches were placed in an arena,
the floor of which was hot (50°C) except
for a small invisible cool area. The wall
of the arena was decorated with visual
patterns having specific geometrical
relationships with respect to the hidden
goal. After 3 trials, cockroaches reached
the hidden goal significantly faster with
such cues than in their absence.
Effects of MB lesions on the ability to
reach hidden goal using spatial cues were
tested. Small pieces of aluminum foil were
implanted into preselected brain areas.
Subsequent histology determined the extent
of lesions. In tests using visible goals,
cockroach with bilaterally lesioned MBs
reached the goal as fast as unoperated
cockroaches. In a hidden goal paradigm,
however, the time taken for bilaterally MB
lesioned cockroaches to reach the goal was
significantly longer than in unoperated
cockroaches, suggesting that MBs are
involved in spatial memory processing.
DISTRIBUTION AND PHEROMONAL RESPONSES
OF DESCENDING NEURONS IN THE BRAIN OF THE
MALE SILKWORM MOTH.
R.Kanzaki, A.Ikeda and T.Shibuya. Inst. of Biol. Sci.
Univ. of Tsukuba, Tsukuba
Male moths, Bombyx mori, display oriented zigzagging
walking in response to the female's sex-attractant
pheromone. Long-lasting excitation (LLE) shown by
brain neurons which descend the ventral nerve cord
(VNC) to the thoracic motor system (descending
neurons, DNs) is one of the important signal to control
the zigzagging behavior.
To study the physiology and distribution of DNs in
the brain of the moth, olfactory responses were
recorded extracellularly from a split off section of one of
the small bundles of the VNC between the brain and the
thoracic ganglion. DNs contained in the bundle were
stained with cobalt from the cut end of the bundle. DNs
were mainly classified into 3 groups (LIL, III) according
to the position of the cell body. DNs of group I and II
have branches in the lateral accessory lobe (LAL) where
many olfactory protocerebral neurons innervate. LLE or
‘Flip-flop (FF)' activities were typically recorded from a
split off dorsal section of a small bundle of the VNC. FF
activities recorded from left and right VNC had an anti-
phase relationship.
Our results suggest that these anti-phase FF activities
to the motor center carried by group I or II DNs may be
important as a zigzag-turning signal of B.mori males.
THE LOCUS OF THE CIRCADIAN PACEMAKER IN
LOCOMOTOR ACTIVITY OF THE HAGFISH,
EPTATRETUS BURGERI.
S. Ooka-Souda!, T. Kadota2, and H.
Kabasawa Atomigakuen Jr. College,
Tokyo, 2Dept. of Anat., Yokohama City
Univ. Sch. of Med., Yokohama, 3aburatsubo
Marine Park Aquarium, Miura.
In a previous study, we found that a
circadian pacemaker may exist in the
anterior part of the hypothalmus of the
hagfish. To determine the detailed
location of the circadian pacemaker, we
recorded the locomotor activity rhythm of
hagfishes after transecting the brain with
scissors and after partial destruction of
brain structures with a high-frequency
lesion generator (tip 0.75 mm). The cuts
and the lesions were made rostral to, in
the center of, and caudal to the anterior
habenula after exposure of the brain
surface. From histological examination of
the location of the cuts and lesions, we
postulated that the pacemaker might exist
in the preoptic nuclei, which have
retinofugal connections as shown by tracer
experiments.
1226 Physiology
GABA,-ERGIC MODULATION OF THE SPINAL
LOCOMOTOR NETWORK IN THE LAMPREY.
T. Matsushima’, J. Tegner’, A. El Manira?, R.H. Hill?
and S. Grillner’.
' Life Sci. Inst., Sophia Univ., Tokyo. * Nobel Inst. for
Neurophysiol., Karolinska _Inst., Stockholm.
GABA plays an important role in the presynaptic inhibition in the
spinal cord. Furthermore, GABA acts, particularly through the
type B receptors, as a modulator of the somato-dendritic
membrane properties of locomotor network neurons.
Enhancement of endogenously released GABA by an
uptake blocker (nipecotic acid) significantly reduced, in a dose-
and time-dependent manner, the burst frequency of the NMDA-
induced fictive locomotion in the lamprey spinal cord in vitro. The
reduction was counteracted by addition of GABA,-antagonists
(phaclofen or 2-(OH)-saclofen), while addition of a GABA,-
antagonist (bicuculline methiodide) caused irregular activities. A
GABA,-agonist (baclofen; 5-404M) alone reduced, and an
antagonist alone increased, the burst frequency in a dose-
dependent manner. Therefore, GABA should be released from
its propriospinal sources during fictive locomotion, and
continuously down-regulating the rhythmic activity through the
GABA, receptors.
At the cellular level, in the lateral gray matter neurons
(intemeurons and motoneurons), baclofen (at the same range of
concentration) suppressed voltage-dependent Ca**-currents (both
HVA and LVA), and concomitantly the following Ca**-dependent
K*-current. Furthermore, current-induced sub-threshold
depolarization and rebound excitation were also suppressed by
baclofen. On the other hand, baclofen brought about no
detectable changes in the resting membrane potential, the
membrane resistance, nor in the threshold level for Na*—action
potentials. Voltage-dependent K*-currents (delayed rectifier and
“A-current") and the inward current through the NMDA
receptor/channels were not influenced.
Modulatory effects of GABA could occur complementarily at
both of the pre- and the postsynaptic sites, which could account
for the GABA, actions in the spinal locomotor network.
IONIC MECHANISMS UNDERLYING ENDOGENOUS
PACEMAKER POTENTIALS OF TERMINAL NERVE-
GNRH CELLS: A CURRENT CLAMP ANALYSIS.
Y. Oka, Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo
Gonadotropin-releasing hormone (GnRH)-immunoreactive
terminal nerve cells have endogenous regular beating discharge
mode, which may be related to their putative neuromodulator
functions. Ionic mechanism underlying the pacemaker potentials
was studied using intracellular current clamp recordings from
whole brain in vitro preparation of a small fish brain. Addition
of 1.5-3 uM TTYX to the Krebs-Ringer solution blocked
spontaneous spikes, but small subthreshold membrane potential
oscillations (pacemaker potentials) remained. About one third of
the recorded cells showed only pacemaker potentials without
spikes in normal perfusing solution. Hyperpolarizing DC current
injections increased the amplitude and decreased the frequency
of pacemaker potentials, and vice versa. Input resistances during
both nsing and falling phases of pacemaker potentials were
smaller than during the resting phase. Thus, sequential
activation of inward (Na+ and/or Ca?+) and then outward (K+)
current is suggested to underlie the pacemaker potentials. The
pacemaker potentials were not blocked by 1mM Ni?+ or ImM
amiloride (blockers of LVA-Ca?+ current) or 2mM Co?*
(blocker of HVA-Ca?* current) or in Ca?+-free solution. On the
contrary, they were blocked by substituting choline or TMA for
Nat. They were also blocked by 5mM TEA, and the resting
potential was raised to a depolarized level. The present results
Suggest that the TIX-resistant persistent Na* current and the
delayed rectifier K* current mainly contribute to the rising and
falling phase of pacemaker potentials, respectively.
THE EFFECT OF CYSTEAMINE ON THE LOCOMOTOR
ACTIVITY RHYTHMS OF THE RAT
C. Fukuhara’, S-I.T. Inouye~, K.Aokil. tLife
eit Inst. Sophia Univ., Tokyo and
Mitsubishi Kasei Inst. of “litemtescacn,
Tokyo
The suprachiasmatic nucleus (SCN) of
the hypothalamus contains a circadian
pacemaker. The dorsomedial SCN is
characterized, in part, by dense
somatostatin-like immunoreactivity CSS=
LI), ee ES functional significance
in circadian pacemaker remains unclear.
A previous study revealed that 24 h SS-LI
contents of the SCN in rats kept under LD
and free-running conditions showed a
distinct circadian rhythm. These findings
suggest that SS levels in the SCN are
controlled by the endogenous pacemaker.
In the present study, we examined effects
of Cysteamine, a SS depletor, on the
locomotor activity rhythms of the rat, to
explore a possible role of SS in the SCN.
Cysteamine (200mg/Kg, I. PD induced
permanent phase shift in subsequent free-
running rhythms in rats. Administration
of Cysteamine reduced SS levels in the SCN
within 5 min after injection and similar
reduction of SS levels was observed also
72. hrs after the injection
The results of this study indicate that
changes in the SS contents in the SCN
induce permanent phase shifts of the
locomotor activity rhythms of the rat.
A NOVEL LEUCOPHORE IN THE DARKBANDED
ROCKFISH, SEBASTES INERMIS is
ULTRASTRUCTURE.
T. Iga and A. Matsuno. Dept. of Biol.,
Shimane Univ., Matsue.
Reflecting chromatophores in the
integument of the darkbanded rockfish,
Sebastes inermis, are of two distinct
types, iridophores and leucophore-like
chromatophores. The iridophores are
smaller and non-motile, producing a
metalic iridescent color. The organelles
involved in the coloration of iridophores
are reflecting platelets. The leucophore-
like cells are larger and highly
dendritic. These cells appear dull brown
by transmitted light, by reflecting light
they are yellowish in appearance. These
cells responded to K and norepinephrine
with pigment dispersion. The response was
opposite to that of melanophores. The
rate of the movements was markedly slow
so that the full dispersion took 20-30
min. The ultrastructure of the
leucophore-like cells was examined. Their
Pigment granules are spherical in form,
varying from 0.8-1.0 pm in diameter. They
contain some fuzzy amorphous substance.
Numerous intermediate filaments were
observed, but microtubules were rather
scarece. From these observations, these
cells were recognized as a type of
leucophores.
Physiology 1227
CYTOSKELETON AND ITS ELEMENTS OF CULTURED
IRIDOPHORES FROM FRESHWATER GOBY,
ODONTOBUTIS OBSCURA
H.Ishida and T.Iga. Department of Biology,
Faculty of Science, Shimane University,
Matsue 690.
Iridophores of the freshwater
tereost, Odontobutis obscura, are motile.
The motility of the iridophores involves
the intracellular translocation of
reflecting platelets. The mechanisms of
the movement of platelets remains to make
clear. In order to reveal the mechanism,
the cytoskeleton and its elements were
examined by using the cultured
iridophores. The cytoskeletons were
exposed by treatment with the zwitterionic
detergent CHAPS and then the iridophores
were fixed with paraformaldehyde. One of
these preparations was observed with
scanning electron microscope. The
cytoskeleton of the iridophore was
composed of a filamentous system with
reticular structure. The other group of
preparations was treated with Rhodamin-
phalloidin, anti-actin antibody, anti-
tubulin antibody or antibody of the other
cytoskeletal elements. Fluorescence
microscopy revealed the reticular
structure of actin filaments through the
cytoplasm. The micro-tubular system was
not observed.
MOTILE MECHANISM OF THE REFLECTING PLATE-
LETS WITHIN THE NEON TETRA IRIDOPHORES.
H. Nagaishi and N. Oshima. Dept. of
Biomolecul. Sci., Fac. of Sci., Toho Univ.,
Funabashi.
(SS EE ESE
Changes in the interference color re-
flected from the motile iridophores, which
are distributed in the lateral stripe skin
of the neon tetra (Paracheirodon innesi),
may be caused by the change in the angle of
inclination of the reflecting platelets in
the cells (theory of venetian blinds,
Nagaishi et al., 1990). Within the irido-
phores, microtubules and both thin and
thick filaments are present, and these
organelles are thought to be related to the
movement of the platelets.
To determine the organelles which par-
ticipate in the platelet movements, we
investigated the spatial distributions and
profiles of the cytoskeletal components by
the conventional electron microscopy and
immunoelectron microscopy. In the ultra-
thin sections of the iridophores which were
cut almost horizontally, microtubules were
observed nearby the edges of the platelets;
they run parallel or perpendicular to the
longitudinal axis of the cell. By the
immunoelectron microscopy, the presence of
actin in the cortical region and between
the platelets was confirmed. Anti-vimentin
antibodies also responded near the edges of
the platelets. From these results, the
role of these organelles and the mechanism
which produces the motive force for the
platelet movements were discussed.
REFLECTION SPECTRA AND COLOR EVALUATION OF
BUTTERFLY ,WINGS 1 2
M.Akimgpto , H.Namiki -Tanaka, H.
Tabata , and A.Yoshida , Dept. of Biology
ch. of Edu., Univ. of Waseda, Tokyo,
Scientific Research Lab., Nissan Research
Centex, Nissan Motor Co.,Ltd., Yokosuka,
and Biohistory Research Hall, Project
Office, Japan Tabacco, Inc., Tokyo.
Structure and optical properties of wings
of Morpho butterflies are studied, aiming
at clarifying the basic characteristics of
structurelly colored materials. Scanning
electoron micrographs of the wing scales
show that the scales have complicated
lamellae structure, and light interference
due to this structure is the origin of
brilliant blue color of the wings.
Reflection spectra of these wings are
measured at various incident and viewing
angles;they are characterized by (i)strong
anisotropy and (ii)high excitation purity
for perception of color(strong and narrow
reflection band). The measurement of color
is done with spectrophotometers using a
specified and constant geometry. However,
there are many applications where measure—
ments at several or different geometries
are necessary. Standard color evaluation
and specification systems for ordinary
color seem to be insufficient for this
kind of structural color, and new systems
are necessary aS a basis for developing
and utilizing structurally colored
materials.
CHROMATOPHORE PATTERN ON INTEGUMENT OF THE
JAPANESE TREEFROG, Hyla arborea japonica.
Yoshihisa Kamishima
Department of Biology, Faculty of Educa-
tion, Okayama University, Okayama 700
Japanese treefrogs are typical arboreal
frogs in Japan, and show bright green
color on the dorsal skin during the active
season. However, the animals kept at low
temperature or under dim light show vari-
ous degree of coloration from pale gray to
dark brown. They also bear darker spots
and bands on the anterior dorsal and
femoral skin characteristic to the indi-
vidual animal. Chromatophore arrangement
in spot and band area is similar to other
portion, but skin glands distribution in
these area are dispersed. Pigment gran-
ules in melanophores in the spot and band
area showed slightly smaller in diameter
than those in lighter area. When the
excised green skin was treated with adren-
ergic $-receptor agonist, isoproterenol,
it took 100 to 300 min to change to gray,
or to darker colors. Melanophore stimu-
lating hormone caused the cells more rapid
response than the neurogenic agent. All
these agents, however, failed to produce
the prominent dark pattern characteristic
to each animal. This may suggests a pos-
sibility of different responsiveness among
and/or differential neuro-humoral control
over the frog skin melanophores.
1228 Physiology
DEMONSTRATION OF LIGHT-GUIDES IN THE EYE
PHOTOPHORES OF THE JAPANESE FIREFLY SQUID.
IG Gleadall!, J-M Bassot2, M Kawahara?,
(e) Inamura?, Y Kito> and Y Tsukaharal.
lRcars, Sendai; 2cNRS, Paris; 3toyama Med.
Pharm. Univ.; Avyozu Aquarium; Sosaka Univ.
This investigation is part of a wider study
of the counterillumination mechanism of the
firefly squid, Watasenia scintillans.
_. The photophores (bioluminescent organs)
in the skin and below the eyes have a
distal spray of straight, membranous
tubules. The function of these tubules in
intact fresh eye photophores was
investigated by confocal scanning laser
microscopy (Bio-Rad MRC 600 system; 488 nm
laser). lue fluorescence emitted from
within the photophore produced an annulus
of light points at the photophore surface.
When the focal plane was changed. to
progressivel deeper locations ('optical
sectioning' the light points were no
longer in focus. The diameter of the
annulus remained constant but the images
became progressively dimmer. This
demonstrates that light is released (and
stimulating light enters) only at the
distal tips of the membranous tubules,
which are therefore naturally occurring
cite eh eee Their function in counter-
illumination may be to ensure uniform
emission of photophore light in a strictly
defined, ventrally directed cone
corresponding to that within which the
squid's body would otherwise be silhouetted
against downwelling sunlight.
THE ROLE OF THE LATERAL EYES AND PINEAL
BODY IN THE COLOUR CHANGE OF THE FRESH-
WATERFISH, ZACCO TEMMINCKI.
I. Takabatake, T. Saiki and T. Iga. Dept.
Biol. Fac. Sci., Shimane Univ., Matsue.
Physiological colour change of Zacco
temmincki, in common with all other tele-
osts, displays a diurnal rhythm. In this
experiment, the effects of eyes and pineal
elimination on the day-night colour changes
OceeZee temmincki were investigated, and
melanophore dispersing and aggregating
activity of blood extract fractionated by
HPLC were examined. In the 12L:12D experi-
ments, blinding caused slight darkening
response in the dorsal melanophores, as
compared with intact fish, during the dark
period. Melatonin contents in the blood
during dark period were relatively in-
hibited by blinding, while in lightness a
rise of blood MSH-like substance was ob-
served. Pinealectomy, in the 12L:12D ex-
periment, did not affect the colour changes
during the light and dark period, but in
the 24DD experiment pinealectomized fish
showed darkening response in contrast with
normal fish. Pinealectomy caused the syn-
thesis of the MCH-like substance during the
dark portion of the photoperiod , while in
the 24DD the synthesis of MSH-like sub-
stance was activated. Pinealectomy of blind
fish caused darkening response during both
light and dark periods. The blood extract
showed the existence of MSH-like substance.
BETA-ADRENERGIC RECEPTOR SUBTYPES OF
MELANOPHORES IN MARINE GOBIES.
H. Katayama. Mukaishima Mar. Biol. Lab.,
Fac. Sci., Hiroshima Univ., Mukaishima-cho,
Mitsugi-gun, Hiroshima-ken.
Previously we reported that the melano-
phore of an estuarine goby, Tridentiger
obscurus possesses both beta-1 and beta-2
adrenergic receptors. In further confirma-
tion of the previous results, I examined
beta-adrenergic receptor subtypes of melan-
ophores of two species of marine goby,
Tridentiger trigonocephalus and Chasmich-
thys dolichognathus gulosus. Denervated
melanophores in split pieces of isolated
caudal fins were used. The aggregation of
pigment was evoked within the melanophores
by verapamil or melatonin in the presence
of phentolamine. Effects of several beta-
agonists on the melanophores in the pres-
ence and absence of beta-antagonist were
photoelectrically recorded. The beta-ago-
nists, adrenaline, noradrenaline, salbuta-
mol and dobutamine, were all effective in
evoking the dispersion of pigment within
the cells. The effects of the beta-agonists
were inhibited by beta-antagonists, meto-
prolol, butoxamine, ICI118551 and proprano-
lol. From graphical analyses of the results
it was estimated that melanophores of each
goby possess both beta-1l and beta-2 adren-
ergic receptors, and that the ratio of
beta-1l to beta-2 receptors in number is
approximately 5:95 in the melanophore of
T. trigonocephalus, and 30:70 in the mel-
anophore of C. d. gulosus.
ADP-RIBOSYLATION OF G PROTEINS BY BACTERIAL
TOXINS INHIBITS THE LIGHT-INDUCED MELANO-
PHORE RESPONSE OF THE XENOPUS TADPOLE.
Y. Miyashita, T. Moriya, K. Asami, N. Yokg-
sawa, S. Hatta, J. Arai~ and _S. Kusunoki~.
Sapporo Med. Coll. 25apporo, IMEC Lab. Dai-
kin Inc., Tsukuba, “LSL Co., Ltd., Tokyo
Pertussis toxin, botulinum exoenzyme C3
and cholera toxin inhibited light-induced
melanin aggregation in melanophores of the
isolated tail fin of the Xenopus tadpole.
The substrate proteins, about 26kDa, 40kDa
and 42kDa, for ADP-ribosyltransferase of
C3, pertussis toxin and cholera toxin, res-
pectively, were present in the tail fin in-
cluding photosensitive melanophores. The
ADP-ribosylation of the proteins by C3 or
pertussis toxin was partially inhibited by
illumination, and the inhibition could be
reversed by addition of guanine nucleotides
In contrast, ADP-ribosylation by cholera
toxin was independent of light. Furthermore,
several kinds of GTP binding proteins(about
55kDa, 42kDa, 34kDa, 26kDa and 23kDa) were
present in the tail fin. From the immuno-
blotting for G proteins, the existence of
Gs, Gi(or Gt) and Gq was confirmed.
These findings suggest that the hetero-
trimeric G protein close to Gi (or Gt) and
the C3 substrate small molecular G protein
interact with a photo-receptive substance
and contribute to its signal transduction
in light-induced melanin aggregation of
the Xenopus melanophores. The substrate of
cholera toxin might be Gs protein and the
toxin appeared to induce melanin-dispersion
by increasing cellular cAMP.
Physiology 1229
ROLE OF NEURAL PROCESSES IN BACKGROUND
ADAPTATION IN THE MEDAKA, ORYZIAS LATIPES.
M. Sugimoto, N. Oshima and R. Fujii. Dept.
of Biomolecul. Sci., Fac. of Sci., Toho
Univ., Funabashi.
In the wild type medaka, a prolonged
background adaptation consists of
Physiological and morphological color
changes. We previously reported that an
adaptation of the medaka to a black (B
fish) or white background (W fish) for 10
days induced an apparent increase or
decrease in the number and the size of
melanophores in the scales, respectively.
The melanophores in B and W fish also
differed in the sensitivity to the
neurotransmitter and hormone. In the
present study, we have examined to what
extent the nervous system affects such
prolonged background adaptation, using
chemically sympathectomized medaka.
The fish was injected with 6-
hydroxydopamine intraperitoneally in order
to denervate melanophores and adapted to a
black (DB fish) or a white background (DW
fish) for 10 days. Melanophore
responsiveness in both DB and DW fish
showed the same tendency to that in B
fish. Melanophores of DB fish were larger
than those of DW fish, although there was
no difference in the number of the cells.
These results indicate that, during the
background adaptation, neural processes
affect the melanophore sensitivity not
only to neurotransmitter but also to
hormone, and have some influences on the
number of melanophores,
SIMULTANEOUS RECORDING OF THE CHANGES IN
INTRACELLULAR LEVELS OF CA“* AND THE MOTILE
RESPONSES OF MELANOPHORES IN TILAPIA.
J. Toyohara and R. Fujii. Dept. of Bio-
molecul. Sci., Fac. of Sci., Toho Univ.,
Funabashi.
REALE AS US a et
By using a handmade dual-wavelength
BO ae fluorometer, which enabled us
eae simultaneously the changes in
fos and the motile response of a
singe ,! ees loaded melanophore, the role
re |(eny in signaling the translocation
of DSpace sense within tilapia (Oreochromis
niloticus) melanophores was investigated.
When the aggregation of pigment was induced
by sympathetic stimulation oF by alpha,
agonists, an increase in [Ca“?t] always
(esos vappea with the motile peers
[Ca appeared highest around the mass of
meee end pigment, and showed a little
elevation in the periphery of the cellular
projections. Mimicking norepinephrine, an
ionophore, ionomycin, induced the elevation
of [Ca“*], as well as the aggregation of
pigment. An alpha, agonist, phenylephrine,
induced pigment aggzegation, but the effect
was abolished in Ca**-free saline solution,
in which [Ca*t]. was normally decreased.
Even in the Ca*~+t-free saline, however,
Sela ie te gradually restored the level
of [cay] ; to the basal level. Presumably,
2 {L@z *]j-mobjlizing mechanism, such as
IP3-induced Ca liberation, is operating.
renee data indicate that the in¢rease in
[Ca is involved in the regulation of
GHEE aggregation in melanophores.
PHARMACOLOGICAL PROFILES OF THE SUBTYPES OF
MUSCARINIC CHOLINOCEPTORS THAT MEDIATE
PIGMENT AGGREGATION WITHIN MELANOPHORES OF
THE MAILED CATFISH, CORYDORAS PALEATUS.
H. Hayashi and R. Fujii. Dept. of Bio-
meolecwil- Sets, PE GE Seila, Welerey Whales,
Funabashi.
Pigment movements in melanophores of
teleost fishes are mainly under the
regulation of the sympathetic nervous
system. Although nerves involved in the
peripheral transmission to the melanophores
in several catfish species, e.g., Corydoras
(Callichthyidae), Pimelodella (Pimelodidae)
and Mystus (Bagridae), are normally adren-
ergic, these fishes possess extra musca-
rinic cholinoceptors which also mediate
pigment aggregation. In this experiment,
using some selective antagonists, we tried
to characterized the cholinoceptor subtypes
mediating aggregation of pigment in the
melanophores of the mailed catfish, Cory-
doras paleatus. Among some cholinoceptor
antagonists, 4-DAMP, a muscarinic M
selective antagonist, most effectively
blocked the pigment aggregating action of
acetylcholine. The results indicated that
the pigment aggregation induced by the
stimulation of muscarinic cholinoceptors
on Corydoras melanophores is mediated by M3
subtype receptors. These observations
suggest that intracellular mechanisms in
the melanophores of this species are
different from those in other teleosts.
Namely, the inositol trisphosphate system
may be involved in this process.
CHANGES IN INOSITOL-PHOSPHATE LEVEL IN THE
FISH MELANOPHORES INDUCED BY NOREPINEPHRINE
F.Morishita, A.Shimada, M.Fujimoto and
K.Yamada. Zool. Inst., Fac. Sci., Hiroshima
Univ., Higashi-Hiroshima.
To examine the second-messenger system
mediating the pigment movement in fish
melanophores, effect of norepinephrine on
the inositol-phosphate level was investi-
gated. For this purpose, the cultured
melanophores of black-moor goldfish, Caras-
sius auratus, were pre-labeled with [?H]-
myo-inositol, and inositol phosphates
extracted were separated by strong-anion
exchange column. Norepinephrine at 100 nM
transiently increased the radioactivity in
inositol triphosphate and inositol tetra-
phosphate fractions to 2-4 fold of control
level. Alpha-antagonists, prazosin and
yohimbine, reduced the NE-effect. When the
cells were pretreated with 1 pg/ml pertus-
sis toxin for 15 hr, the NE-induced in-
crease of inositol phosphates was attenuat-
ed, suggesting the involvement of GTP-
binding protein in the cell response. The
lipid analysis by thin-layer chromatography
indicated that the increase of inositol
phosphates was accompanied by decrease of
the radioactivity in inositol phospho-
lipids.
These results suggest that stimulation
of alpha-adrenoceptors increases the
inositol-phosphate level in fish melano-
phores.
1230 Physiology
CHICKEN VISUAL PIGMENTS AND COLOR VISION
T. Yoshizawa. Department of Applied Physics
and Chemistry, The University of Electro-
Communications, Chofu.
Recently we showed that chicken iodopsin
is in both principal and accessory members
of double cone with or without oil droplet
(green), respectively, and in a single cone
with a red oil droplet, and that chicken
green, blue and violet are located at
single cones with yellow, clear and blue
oil droplets, respectively. Since incident
light to these cone pigments passes through
the inner segment with or without an oil
droplet, it acts like a cut-off filter.
The spectrum of iodopsin was corrected
with the transmittance curve of green oil
droplet or the corresponding position in
the principal or accessory member, respec-
tively. Both the corrected spectra are so
close to chicken photopic sensitivity curve
that the double cone would be responsible
for light intensity discrimination in
daylight vision. The spectra of chicken
red, green, and blue were corrected by
transmittance curves of the respective oil
droplets, resulting in remarkable decrease
of overlaps of two spectra between chicken
red and blue and between chicken green and
violet. Thus almost all the wavelengths
between 400 and 600 nm can be discriminated
with ratios between absorptions by only two
types of cone pigments. This would result
in increase of the resolution of wavelength
discrimination.
PORPHYROPSIN AND NEW DEEP-SEA VISUAL
PIGMENT WITH 4-HYDROXYRETINAL ARE FOUND IN
SOME MESOPELAGIC CEPHALOPODS IN ATLANTIC.
Y.Kito! K.Narita M.Seidout M. Michinomae2
iG Partridge’and Pod) a Herring? lpept. of
Biol., Osaka Univ., Toyonaka,“Dept., of
Biol., Konan Univ., Kobe,~Dept.of Zool.,
Univ. of Bristol, Bristol, ‘Inst. of Ocean.
Sci., Deacon Lab., Surrey.
Firefly squid, Watasenia scintillans,
has three visual pigments in its retina,
each pigment being segregated in different
parts and based on ditferent chromophore,
i.e., Al-based pigment (Amax: 484nm), A2-
based pigment (Amax:500nm) and A4 pigment
based on 4-hydroxyretinal (Amax:470 nm).
We found that among cephalopods, collected
by R/V Discovery from Atlantic, squids,
Pyroteuthis, Pterigioteuthis, and octopus,
Japetella had two visual pigments with Al
and A4, and squid, Bathyteuthis had three
pigments with Al, A2 and A4. This
indicates another molecular adaptation of
visual pigment to photic environment. The
hypsochromic shift of absorbance spectrum
is due to solvent effect in the nonpolar
environment of the retinal-binding site
of opsin. The Schiff base compound of
4-hydroxyretinal showed the same effect.
HPLC ANALYSIS OF RETINOIDS EXTRACTED
FROM THE_ PLANARIAN, Dugesia japonica.
K. Azuma N.Iwasaki-, M.Azuma~“, ve
Shinoz wae M. Ezaki~, S.Nakamura~ and T.
Suzuki~. \oept. of Biol., Osaka Med.
Coll., Takatsuki, “Dept. oe Health Sci.,
Osaka Kyoiku Univ. Osaka, ~Dept. of biol.
and chem. Engin,, Fac. of Engin. Univ. of
Gunma, Kiryu, Dept. of Biol., Fac. of
pele; Univ. of Hirosaki, Hirosaki and
Dept. of Pharmacol., Hyogo Coll. of
Med., Nishinomiya,
Retinal and retinol (including retinyl
ester) were extracted from the planarian
bodies and analyzed by HPLC. 11-Cis
retinal was only detected in the extracts
from the head piece (anterior part
containing eyes). The amounts of all-
trans retinal, 11l-cis retinal and all-
trans retinol including the retinyl ester
were O.1-1.1, 0.11-0.19, and 20-50
pmol/head, respectively. Almost all of
all-trans retinol and the retinyl ester
were extracted with hexane from the
freeze-dried head pieces, but all-trans
and 11l-cis retinals were not. These
retinals were extracted from the residues
after the hexane extraction by the oxime
method. These results suggest that all-
trans retinal is bound to an unidentified
protein in the tissues and 1l-cis retinal
exists as the chromophore of the visual
pigment in the eye.
PHOTOBLEACHING PROCESSES OF GECKO VISUAL
PIGMENTS.
D.Kojimal, T.Okanol, H.Imail, Y.Fukadal,
Y.Shichidal, F.Crescitelli2 and
T.Yoshizawa?. IDept. of Biophys., Fac. of
Sci., Kyoto Univ., Kyoto, 2Dept . of Biol,
Univ. of California, LA, U.S.A. and 3 Dept.
of Appl. Phys. and Chem., Univ. of Electro-
Communications, Chofu.
The Tokay gecko (Gekko gekko), a noctur-
nal lizard, has two kinds of visual
pigments, P521 and P467. In spite of the
pure-rod morphology of the photoreceptor
cells, amino acid sequences of P521 and P467
are the most similar to the typical cone
visual pigments, iodopsin and chicken green-
sensitive cone visual pigment, respectively.
In order to get further evidence that the
gecko visual pigments are really the cone-
type pigments, we investigated their
photobleaching processes by means of low
temperature spectrophotometry.
P521 has a similar bleaching process to
iodopsin. However, unlike batho-
intermediate of iodopsin, that of P521
mainly converts to the next intermediate.
The meta-intermediates of both of P521 and
P467 are thermally less stable than those of
rhodopsin. Since the less stabilities of
meta-intermediates are one of the characters
of cone visual pigments distinguishable from
rod visual pigments, we can safely concluded
that both of gecko visual pigments have
functional properties of cone-type visual
pigments.
Physiology 1231
THE ABSOLUTE STRUCTURE OF 3-HYDROXYRETINAL
USED AS THE VISUAL PIGMENT CHROMOPHORE IN
INSECTA.
T.Seki!', K.Isono® and M.Ito%. ‘Dept. of
Health Sci., Osaka Kyoiku Univ., Osaka,
?RCAIS, Tohoku Univ., Sendai, and *Kobe
Women's College of Pharmacy, Kobe.
In the compound eyes of insects, two
kinds of retinal congeners, retinal (ret, )
and 3-hydroxyretinal (ret,;), are used as
the visual pigment chromophore. Being
different from ret,, the ret, is a chiral
molecule, and so the absolute structure of
ret, in the eyes of fruitfly, butterfly,
cicada and dragonfly was determined.
Retinoids in the compound eyes of
insects were extracted by the HCHO-method,
and the peaks of all-trans and 11-cis ret,
on HPLC were collected. The 171-cis
fraction, which should have come from the
visual pigment chromophore, was isomerized
by exposure to white light, rechromato-
graphed, and the resulting all-trans ret,
was collected. The absolute structure of
both the original all-trans and the photo-
isomerized all-trans (originated from 11-
cis) ret; was determined by HPLC using a
chiral column (ChiraSpher, Merck) ‘!) .
In the case of fruitfly, 11-cis ret, was
exclusively (3S), but the original all-
trans ret; was dominantly (3S) containing
(SR) at the ratio of 10-20%. In the
compound eyes of butterfly, cicada and
dragonfly, by contrast, only the (3R) -all-
trans and (3R)-11-cis enantiomers were
detected ae
1: Ito et al, J. Nutr. Sci. Vitaminol.
38, 111-115 (1992) .
OPSIN PROCESSING IN THE PROTEIN-SYNTHESIZ-
ING PATHWAY OF DROSOPHILA PHOTORECEPTOR
CELLS.
K. Ozaki, H. Nagatani, M. Ozaki and F. Tokunaga.
Dept. of Biol., Fac. of Sci., Osaka Univ., Toyonaka.
The synthesis of Drosophila opsin is promoted by its
chromophore, 11—cis—3—hydroxyretinal. When flies are
raised on a carotenoid—free medium, no mature opsin (35 kD)
is formed in their retina, but a trace amount of opsin precursor
(40 kD) is accumulated. This opsin precursor possesses an
oligomannose-type sugar chain, which is completely digested
during the maturation after chromophore—binding.
When flies are irradiated with blue light, a major part of
main rhodopsin (Rh1) is converted to metarhodopsin, and the
prolonged depolarizing afterpotential (PDA) is evoked in their
photoreceptor cells. Under such condition, the photoreceptor
cells synthesized 37 kD opsin instead of mature 35 kD opsin.
The apparent molecular mass of the 37 kD opsin was reduced
to 35 kD by the treatment with peptide N—glycosidase F, while
it was not changed by the treatment with endoglycosidase H.
This result suggests that 37 kD opsin has a sugar chain which
has been partially processed in the golgi apparatus of photore—
ceptor cells.
The norpA mutant, which bears mutation on an eye-specific
phospholipase C gene, possesses enough rhodopsin but fails to
evoke receptor potential. In this mutant, mature 35 kD opsin
was synthesized after blue-irradiation. Moreover, even in the
wild-type flies, mature 35 kD opsin was synthesized when red
or green light were given just after blue—irradiation.
On the basis of the above data, we concluded that the 37 kD
opsin is the metabolic intermediate sitting between 40 kD
precursor of opsin and mature 35 kD opsin. In addition, it was
suggested that the opsin processing could be modulated with
the activation of phospholipase C, or the changes in intracellu—
lar ionic condition caused by cell excitation.
IN VITRO SYNTHESIS OF DROSOPHILA OPSIN.
K. Katanosaka, F. Tokunaga and K. Ozaki.
Dept. of Biol., Fac. of Sci., Osaka Univ., Toyonaka.
Drosophila opsin (Rh1) has two potential glycosylation sites
at Asn-20 and Asn-196. Although the precursor of opsin is
actually glycosylated, mature opsin has no detectable oligo-
saccharide chain. This fact thus suggests that the oligosaccha—
ride chain is eliminated during the maturation of opsin.
In order to examine the process of opsin maturation, we
synthesized the opsin in a cell—free translation system using
reticulocyte lysate. In the absence of pancreatic microsomal
membranes, only 36k opsin was synthesized. On the contrary,
three kinds of opsin with different MW (36k, 40k, 43k) in
SDS-PAGE were detected when the synthesis was carried out
in the presence of the microsomal membranes. Digestion of
N-linked oligosaccharides with peptide N-glycosidase F
(PNGase F) reduced the apparent MW of 40k and 43k opsins
to 36k, indicating that both 40k and 43k opsins are glycosylat-
ed, while 36k opsin is not. This behavior of 40k opsin was
quite similar to that of the opsin precursor found in vivo.
We next analyzed the time course of in vitro opsin synthesis.
In the early stage of the reaction, 43k molecule was the domi-
nant member of the glycosylated opsins, while the fraction of
40k opsin gradually increased later. In order to check the
possibility that the change in MW from 43k to 40k caused by
the partial trimming of the oligosaccharide chain, we blocked
the initial step of the trimming with N-methyldeoxynojirimy—
cin. The result that the untrimmed opsin has MW of 41k,
however, suggested that the decrease in MW from 43k to 40k
results from complex reactions including the usual trimming
of oligosaccharide chain. Considering the extent of MW shift
by the PNGase F treatment, it was also suggested that 40k
opsin synthesized in vitro (and probably 40k opsin precursor
in vivo, too) possesses a Slightly trimmed oligosaccharide
chain.
EXPRESSION OF VISUAL PIGMENTS IN MAMMALIAN
CELLS.
F. Tokunaga and O. Hisatomi
Dept. of Biol., Fac. of Sci., Osaka Univ.,
Osaka.
Lamprey is one of the most primitive
vertebrates. The outer segment of the
short photoreceptor cell of lamprey is cone
type. The ceo, however, contains
rhodopsin-like visual pigment (lamprey
rhodopsin). We isolated a cDNA of
lamprey visual pigment. The deduced
amino acid sequence showed >75% identities
with those of rhodopsins of higher
vertebrates.
The cloned cDNA was inserted into an
expression vector and transfected into the
mammalian cells by using calcium phosphate
method. After 24-48 hr, the cells were
harvested and the membrane fraction was
isolated by sucrose flotation. The
purified membrane fraction was mixed with
1li-cis retinal, and visual pigments were
extracted with 2% CHAPS. By photo-
bleaching experiments, we observed a
reconstituted pigment with difference
maximum at about 500 nm, which coincides
with that of lamprey rhodopsin isolated
from the lamprey retina. The expressed
pigment, as well as lamprey rhodopsin,
bleached gradually in the presence of 100
mM NH,OH in the dark. These results
indicate that the cloned cDNA encodes
lamprey rhodopsin, and that the expressed
pigment can be used as lamprey rhodopsin.
1232 Physiology
Distribution of opsin in the photoreceptors in the late
pupal and newly emerged Drosophila
Atsuko Matsushita, Kentaro Arikawa, and Eisuke Eguchi.
Department of Biology, Yokohama City University,
22-2 Seto, Kanazawa-ku, Yokohama 236, Japan.
Change of opsin distribution in the course of the development
of rhabdomeric photoreceptors was examined in the late pupal
and newly emerged Drosophila by electron-microscopic
immunogold labeling.
Numerous gold particles that represent the localization of
opsin were found in the rhabdomeres of control flies (Canton-S)
at 2 days before emergence, but were not found in the flies of 3
days before emergence, the time when the rhabdomeric
microvilli begin to develop.
Besides the microvilli, the immunoreactivity appeared in the
multivesicular bodies, rough ER, and secondary lysosomes as
previously reported by others. In addition, the labeling was
found on some membrane-enclosed inclusion that was com-
posed of electron-dense vesicles, lamellated membranes, and
ribosomes. The inclusion is always found in the rER-rich cy-
toplasm, where the protein synthesis takes place at a high rate.
The inclusion first appears at the end of the pupal stage and
disappears within 1 day after emergence: i.e. not found in the
adult older than 1 day. The inclusion was not found in a mutant
DPP- in which the development of the rhabdomeric microvilli
stops in the late pupal stage.
According to the accepted interpretation of the membrane
turnover process, the inclusion described here should be cate-
gorized as the secondary lysosome. However, the present re-
sults indicate that the inclusion may be a structure for the tem-
poral storage of over-synthesized rhodopsin, or, perhaps a
structure that is involved in the synthetic pathway of the
thabdomeric membrane.
The antiopsin is a generous gift of Dr. T. Tanimura.
Iodopsin immunoreactivity in retina and
pineal organ of river lamprey, Lampetra
japonica.
S. Tamotsu?, T. Oishi*, K. Nakao2, Y.
Fukada3, Y. Shichida3, T. Yoshizawa* and
Y. Morita!. list Dept. of Physiol.
Hamamatsu Univ. Sch. of Med., Hamamatsu,
2Dept. of Biology, Faculty of Science,
Nara Women's Univ., Nara, 3Dept. of
Biophysics, Faculty of Science, Kyoto
Univ. Kyoto, *Dept. of Applied Physics
and Chemistry, The Univ. of Electro-
Communications, Chofu.
In the present study retina and pineal
organ of lamprey were investigated
immunocytochemically using an antibody
against the iodopsin, the chicken red-
sensitive cone visual pigment.
In the retina, only outer segments of
the long photoreceptor cells showed
lodopsin-immunoreactivities (I-IR). In
the pineal organ, the I-IR photoreceptors
were observed at both ventral and dorsal
wall, and at the pineal stalk. Further-
more, following four types of pinealocyte
were identified by mean of a double
immunostaining for the iodopsin and the
serotonin; 1)both serotonin- and
iodopsin-immunopositive, 2)serotonin-
immunopositive and iodopsin-immunonega-
tive, 3)reversal to the 2nd type, 4)no
immunoreactivity to both antibodies.
PRESUMPTIVE PHOTORECEPTOR CELLS IN THE
SIPHON OF A CLAM, TAPES (AMYGDALA)
PHILIPPINARUM.
H. Karakisawa and K. Ohtsu. Ushimado Marine
Laboratory, Okayama University, Okayama.
A clam, Tapes (Amygdala) philippinarum
responds to a light stimulus given locally
to the siphon by retracting it and closing
the shells, indicating an existence of
photoreceptor cells in the siphonal region.
Light- and electron-microscopical
observations revealed 4 types of cells,
epithelial cells, muscle cells, nerve cells
and granule-rich cells (granular cells).
Among them, however, no typical photo-
receptor cell could be found. Instead, the
epithelial cells bore striking microvilli
similar to those of molluscan photoreceptor
cells, suggesting that they might have a
photoreceptive function and further
rhodopsin might be found in the microvilli.
To clarify this, an anti-squid(Toderodes
pacificus) rhodopsin serum (mouse) was used
in conjunction with FITC-labelled anti-
mouse IgG. Specific fluorescence was
detected clearly at the microvilli of the
epithelial cells, and at the granular cells
as well. It disappeared, however, by
treating the anti-squid rhodopsin serum
with Toderodes rhodopsin extracted by
digitonin. It is suggested from these facts
that rhodopsin exists in the microvilli of
the epithelial cells as well as in the
granular cells and they may function as the
photoreceptor cells.
ANTI-GECKO OPSIN ANTIBODY RECOGNIZING OUTER
SEGMENTS OF RED-SENSITIVE CONES IN FRESHWATER
TURTLE AND CHICKEN RETINAS
M. Yoshida? and K. Mifune? !Inst.Biol.,Qita Univ.
Oita, 2Dept. Microbiol., Med. College of Oita,
Hazama, Oita.
Monoclonal antibodies (mAbs) were raised to a
suspension of retinal rod outer segments (ROSs)
from geckos, Gekko japonicus. Hybrydomas produc-
ing anti-ROS were screened with indirect immuno-
fluorescence method on cryostat sections of
gecko retinas. The mAbs reacted with bands cor-
responding to opsins as demonstrated by SDS-PAGE
of retinal homogenates solubilized with deter-
gent.The mAbs labeled ROSs of all photorecep-
tors, except for those of one member of type
C double cells. This labelling pattern was fur-
ther confirmed by using the IgG gold-silver
technique at the LM level. Whole-mount prepa-
rations of freshwater turtle and chicken ret-
inas treated with immunohistochemical SAB reac-
tion showed that the mAbs labeled OSs of cones
with red oil droplet and of double cones, but
not those of rods, and of green and blue cones.
The raised mAbs specific for opsins of gecko
rods with P521 pigment seems to recognize com-
mon epitopes in the OSs of red-sensitive cones
in turtle and chicken retinas. In the retinas of
diurnal lizards, Jakydromus tachydromoides,both
OSs of double cones and of cones with yellow
oil droplet were also labeled with the mAbs.
Physiology 1233
LOCALIZATION OF VISUAL PIGMENTS IN THE
VERTEBRATE BRAIN.
Ts Oishil, Y. Yahirol, T. Yoshikawal, H.
Masuda2, Te Yoshizawa3, and M. Michinomae?.
IDept. of Biology, Nara Women's Univ.,
Nara, 2Dept. of Cell Biol., Chest Disease
Res. Inst., Kyoto Univ., Kyoto, 3Dept. of
Biophysics, Kyoto Univ., Kyoto, and 4Dept.
of Biology, Konan Univ., Kobe.
We investigated localization of visual
pigments in the extraretinal photoreceptors
of frogs (Rana catesbeiana) and birds
(Coturnix coturnix japonica) using immuno-
cytochemistry and HPLC.
The outer segments of pinealocytes in
the frog pineal were well stained with
anti-serum against bovine rhodopsin (Rh-As)
and weakly stained with monoclonal
antibodies against chicken iodopsin (Io-
mAb) . There are some immunonegative cells
observed. The cells in the frontal organ
were immunopositive to both Rh-As and Io-
mAb. Paraventricular cells in the hypo-
thalamus were stained with Rh-As, but not
with lIo-mAb. In the pineal of birds,
pinealocytes were immunopositive to Rh-As,
but the reactivity to Io-mAb was very weak
Or none. And much individual variation was
observed. Some cells in the hypo-thalamus
were also stained. HPLC analysis revealed
1l-cis and all trans retinal in the pineal
complex of the two species, and 11l-cis and
all-trans dehydro-retinal were detected in
the pineal of frogs.
THREE-DIMENSIONAL DISTRIBUTION OF PROTEO-
GLYCANS IN THE MOUSE INTERPHOTORECEPTOR
MATRIX.
M. Iwasaki’, A. Tawara2, M. E. Rayborn3 and J. G.
HollyfieldS. 1Biol. Lab., Fac. of Sci., Fukuoka Univ.,
Fukuoka, 2Dept. of Ophthalmol., Fac. of Med., Kyushu
Univ., Fukuoka and 3Cullen Eye Inst., Baylor Col. of
Med., Houston, USA.
The interphotoreceptor matrix, filling the extra-
cellular compartment between the neural retina and the
pigment epithelium in the vertebrate retina, has been
implicated in a variety of functions, including in retinal
attachment, phagocytosis, and metabolite trafficking.
Proteoglycans in the mouse interphotoreceptor matrix
were stained with Cupromeronic blue, and 1pm-thick
sections were visualized by means of Electron
Spectroscopic Imaging (ESI) technique. Analyses of
stereo pairs of ES! showed extensive invagination
between proteoglycan networks and apical processes
of the pigment epithelium. Rinsing of the isolated
Pigment epithelium with Ringer's solution did not
eliminate proteoglycans near the tips of apical
Processes. These results suggest that the insoluble
proteoglycans play a role in retinal attachment.
LIGHT-INDUCED BINDING OF PROTEINS IN THE
CRAYFISH RHABDOMERIC MEMBRANES
(PROCAMBARUS CLARKI/).
A.Terakita*, Y.Tsukahara, T.Hariyama!, T.Seki2 and
H.Tashiro, Photodynamics Res. Cent., RIKEN, 1RCAIS,
Tohoku Univ., Sendai, and 2Dept. of Health Sci., Osaka
Kyoiku Univ., Osaka. * Present address: Fac. of Educ.,
Oita Univ., Oita.
Light-induced protein interaction in the process of the
visual transduction in arthropods photoreceptor was
investigated biochemically using crayfish retina.
Irradiated and non-irradiated rhabdomeric membranes
were incubated with buffer-soluble proteins extracted
from the dark adapted retinas and analyzed by SDS-
PAGE. A 40 kDa and a 46 kDa proteins were found to
bind to the irradiated membranes but not to the non-
irradiated membranes. The light-induced binding of the
proteins was also observed in the intact retina.
The soluble proteins of crayfish cross-reacted with
blowfly rhabdomeric membranes. They were bound to
the membranes with formation of metarhodopsin and
dissociated from the membranes with photoregeneration
of rhodopsin, showing the similar property to the 48 kDa
protein in the fly retina (Bentrop & Paulsen, 1986).
Immunoblot analysis was carried out by using an
antiserum against the mouse arrestin (S-antigen).
The antiserum cross-reacted with the 40 kDa protein but
not with the 46 kDa protein.
These results suggest that the binding of the 40 kDa
and 46 kDa proteins to the membranes is caused by the
formation of metarhodopsin, and the 40 kDa protein has
similar structure to vertebrate arrestin.
MUTATION THAT DISRUPTS RHABDOMERES OF
RETINULAR CELLS NONSPECIFICALLY IN THE
COMPOUND EYES OF DROSOPHILA
K.Isono ,,T. Hariyama A K.Arikaya”, 4
-Komatsu~, D.Yamamoto’, R.Ueda*, Y.Sano”~.
Res. Center for Appl. ing ormalcion
Sci.,Tohoku Univ., Sendai,“Dept. of
Biology, Yokohama City Univ. ,Yokohama,
Dept. of Physiology, Tokyo Wemen’s
College of Medicine, Tokyo and ‘“Mitsubishi-
Kasei Inst.of Life Sci., Machida.
A photoreceptor mutant was isolated
from screening stocks obtained originally
by enhancer-trap insertions.
The mutation, which is tentatively
named ara (all-rhabdomeres absent)
eliminates photosensitivity of all three
classes of the retinular cells in the
compound eyes. EM study revealed that
rhabdomeres are almost absent for all
types of the photoreceptors. Pupal
immature rhabdomere seemed to develop
normally but remained small before
eclosion. Within 24 hours the rhabdomeric
structure undergoes degeneration.
Genetic analysis showed that the gene
is on the distal end of the right arm of
the third chromosome where cht, another
photoreceptor specific gene has been
reported. Complimentation test between
these mutations is in progress.
1234 Physiology
PHOTORECEPTOR DEVELOPMENT IN THE PINEAL
ORGAN AND THE RETINA OF THE SALAMANDER,
Hynobius dunni.
H. Takahama, Biological Institute, Faculty
of Education, Oita University, Oita
Morphological changes in the pineal
and retinal photoreceptors of the salaman-
der, Hynobius dunni, were examined by
scanning electron microscopy after the
eryofracturing and immunoreactivity of
tubulin during development. At stage 39,
the inner segments and budding cilia of
the photoreceptor cells are visible in
both organs. At stage 41, the outer seg-
ments possess whorl-like membranous lamel-
lae in the pineal and retinal photorecep-
tors. At the hatching stage (43), the
outer segments are composed of regular
well-stacked membranous lamellae, and the
inner segments are well-labeled for anti-
tubulin and the reaction products are
oriented along cell axis in the retinal
photoreceptors. On the ohter hand, in the
pineal photoreceptors, the outer segments
possess irregular membranous lamellae and
the inner segments are stained intensively
but randomly for anti-tululin. These
results suggest that the photoreceptors
develop simultaneously in the pineal organ
and retina but maturation of the pineal
photoreceptors is delayed.
SPECTRAL RESPONSES AND STRUCTURE OF
HORIZONTAL CELLS IN THE FILEFISH RETINA
K.Kawamatal, T.Osaka!, K.Ohtsuka!, H.Utiyama?, and
T. Ohtsuka3
iFac. of Educa., Akita Univ., Akita, 2Dept. of Anat., Nippon
Med. Sch., Tokyo, 3Natl. Inst. for Physiol. Sci., Okazaki
We studied the correlation between the physiological and
the morphological properties of the second order neuron in the
retina of the filefish, Novodon modestus. The intracellular
response of the horizontal cell (HC) to the monochromatic
flashes was obtained by a glass microelectrode filled with
biocytin (or Lucyfer yellow) solution, and then the same cell
was injected electrophoretically with the dye. The spectral
responses collected from 103 HCs were classified into three
subtypes; 90 cells hyperpolarized to all the wavelengths of
monochromatic stimuli, 9 cells hyperpolarized to green and
blue flashes, while depolarized to red flashes, and 4 cells
hyperpolarized to both blue and red flashes, while depolarized
to yellow flashes. These HCs are denoted shortly by LHC,
BHC, and THC, respectively. We found all LHCs inthe
filefish have a maximal response at 480 nm, while it has been
repoted that LHCs in the freshwater teleosts have a maximal
response at around 640 nm.
The intracellular dye-injection revealed functional
organization of the horizontal cell layer. (1) LHC, BHC, and
THC are independently coupled probably by gap junction, and
each makes a functional syncytial sheet. (2) In each HC sheet,
we found the somata of three subtype of HCs were arranged in
regular mosaic. (3) The syncytial sheet of LHC consisting of
somata and axon terminals made a distal sublayer of the INL,
while the syncytial sheets of both BHC and THC, lacking
axon terminals, made a proximal sublayer.
POTASSIUM CHANNELS CLOSED BY LIGHT IN THE
ONCHIDIUM EXTRAOCULAR PHOTORECEPTOR.
T. Gotow, T. Nishi and M. Kameyama.
Dept. of Physiol., Sch. of Med.,
Kagoshima Univ., Kagoshima.
Previous studies have shown that the
depolarizing receptor potential (the photo-
response) of an extraocular photoreceptor,
the photoresponsive neuron (A-P-]) in a mol-
lusc, idium results from the light sup-
pression of K currents, flowing during
dark. We report here the single-channel
currents suppressed by light, recorded from
cell-attached patches of A-P-1. The rate
of channel activity was largely unaffected
by de- or hyper-polarizing the patches, in-
dicating that the suppression of channel
activity is caused by light itself but not
by voltage changes. The single-channel
conductance was estimated to be 50-100 pS,
from the slope of the relation between
channel current and patch polarization.
The reversal potential and its shift for
the single channel currents, measured from
patch electrodes filled with different K
solutions suggested that the light-sensi-
tive channels were specifically selective
for K ions. Finally, kinetics of the
light-sensitive channels was investigated.
These results support that the photore-
sponse of the extraocular photoreceptor, A-
P-1 results from the closure of specific
light-sensitive K° channels. The light-
sensitive channel was active in the dark-
adapted A-P-1.
LIGHT ADAPTATION AND CALCIUM FEEDBACK IN
PRIMATE RODS.
K. Nakatani’, T. Tamura? and K-.W. Yau%. ‘Inst. of
Biol. Sciences, Univ. of Tsukuba, Tsukuba, 2Dept. of
Ophthalmology, Kanazawa Univ. Sch. Med.
Kanazawa 3%Dept. Neuroscience, Johns Hopkins Univ.
Sch. Med., Baltimore, USA.
It is now known in amphibians that when retinal rods
are illuminated there is a net efflux of Ca?* in the rod
outer segment. This Ca2* efflux leads to a decrease in
free calcium, which in turn feeds back negatively on
the phototransduction process to regulate the light
sensitivity. Relatively little is known in mammalian
rods. Using a suction pitet to record membrane
current from an isolated primate rod, We examined the
kinetics of the electrogenic Na*-dependent Ca?* efflux
during illumination and found that the rate of this efflux,
which reflects the internal free calcium concentration,
declined with a time constant of about 100msec. In
separate experiments including the removal of this
Ca?* feedback, the response of a primate rod to a dim
flash was found to increase by 2-3 fold, accompanied
by an increase in the time-to-peak of the response.
These changes can be broadly reproduced by a
quantitative model of phototransduction that has the
Ca2*+ feedback as one of its features. Finally, the
adaptation to background light that we have observed
in these cells most likely also arises from the Ca2*
feedback.
Physiology 1235
APPEARANCE OF IMMUNOREACTIVE PHOTORECEPTOR
AND BIPOLAR CELLS IN THE NEWT RETINA
DURING REGENERATION
M.Niino and T.Saito. Inst. of Biol. Sci.
Univ. of Tukuba, Ibaraki
I
It is known that the adult newts possess
the ability to regenerate a functional
retina following the complete removal of
the original retina. The order of appear-
ance of photoreceptor and bipolar cells
during retinal regeneration was studied
immunohistochemically. A mouse monoclonal
antibody (RB-1l), specific for photorecep-
tors (cone) and bipolar cell subtypes in
adult newt, was generated. RB-1l immuno-
reactive photoreceptors appeared in the
regenerating retina before separation of
two plexiform layers. The bipolar cells
appeared shortly after the separation of
the plexiform layer. RB-l-labelled bipolar
cells were characterized by the soma lying
in the middle or slightly distal level in
the inner nuclear layer and the axon
terminal ending at the most proximal level
of the inner plexiform layer (IPL).
Intracellular recording and dye injec-
tion in the retinal cells showed that ON
bipolar cell axons terminate in the inner
half of the IPL, while OFF bipolar cells
terminate either in the outer half or in
the inner half. Combining intracellular
staining techniques with immunohisto-
chemistry suggested that RB-l-labelled
bipolar cells are not ON bipolar cells,
but OFF bipolar cells.
GLUTAMATE RECEPTORS OF SOLITARY SPIKING
CELLS IN THE NORMAL AND REGENERATING NEWT
RETINA.
C.Chiba, H.Sakai and T.Saito. Inst. of
Biol. Sci. Univ. of Tukuba, Ibaraki.
Glutamate receptors of solitary spiking
cells isolated from the newt (Cynops
Pyrrhogaster) retina were studied with
whole-cell patch-clamp techniques. The
solitary spiking cells responded to pres-
sure application of L-glutamate with
either depolarization (80%) or hyperpolar-
ization (10%). The glutamete-induced depo-
larizing response initiated spikes and
hyperpolarizing response inhibited spike
initiation, indicating that there are at
least two types of glutamate receptors,
exitatory and inhibitory, in the spiking
cells. The depolarizing response was
associated with a decrease in input
resistance and produced by inward
Currents. The hyperpolarizing response was
associated with an input resistance
increase and produced by outward currents.
Both inward and outward currents were
reversed their polarity at about O mV.
The adult newts possess the ability to
regenerate a functional retina following
the complete removal of the original
retina. Appearance of glutamate response
during retinal regeneration was studied.
Solitary spiking cells in the regenerating
retina have shown the depolarizing
response to L-glutamate before segregation
into distinct synaptic layers has begun.
MODULATION BY L-GLUTAMATE OF CALCIUM
ACTION POTENTIAL IN DISSOCIATED HORIZONTAL
CELLS FROM CATFISH RETINA.
K. -I. Takahashi and D. R. Copenhagen®. “Dept.
Physiol., Keio Univ. Sch. Med., Tokyo and Depts.
Ophthalmol. & Physiol., UCSF Sch. Med., San Francisco,
CA, U.S.A.
Dissociated horizontal cells produce regenerative
action potentials(APs) when depolarized by currents injected
through conventional microelectrodes. This AP results from
a sustained high voltage—activated(HVA) calcium current
that is activated positive to -40mV. L-glutamate(100uM)
reduces the amplitude and the duration of the AP. Direct
measurement of an intracellular pH([pH];) of the cells with
BCECF-AM reveals that L—glutamate also acidifies in a
dose-dependent manner. If L-glutamate modifies the AP
via [pH];, then independent modification of [pH]; should
similarly affect the AP. Acidification induced by application
of Na-acetate(25mM) and washout of NH,Cl(20mM)
dereased the amplitude and the duration of the AP, and
alkalinization induced by application of NH,Cl exerted the
opposite effects, as hypothesized. These results suggested
that HVA calcium channel activity was suppressed by
acidification of horizontal cells induced by L—glutamate.
Horizontal cells in situ have a dark resting membrane
potential between —40 and -20mV, which is above the
activation voltage for the HVA current. Jn situ,
extinguishing a light stimulus causes a release of L—
glutamate from photoreceptors and depolarizes horizontal
cells from a maximum potential, which can be as high as
-80mV, to the normal dark potential. No sustained APs are
seen under these conditions. We hypothesize that in situ the
regenerative AP is suppressed by acidification induced by
L-glutamate released from photoreceptors.
VISUAL RESPONSE OF NON-SPIKING GIANT
INTERNEURONS IN THE BRAIN OF CRAYFISH
H. FURUDATE, Y. OKADA AND T. YAMAGUCHI. Dept. of
Biol., Fac. of Sci., Okayama Univ., Okayama
There are five pairs of nonspiking giant inter-
neurons (NGIs) in the protocerebrum of the
crayfish. These NGIs have thick and long dendritic
processes extending across the midline and are
post synapticto the fourth order visual
interneurons (SFs). Three pairs of NGIs (G1, G2,
G3) running in a cluster responded with graded
depolarizing and hyperpolarizing potentials to
illumination on total surfaces of the ipsilateral and
contralateral eyes, respectively. Although the
amplitude of hyperpolarizing potentials recorded
from the contralateral side of dendritic process
was always larger than that recorded from the
ipsilateral side, the relationship between the
polarity of potentials and the recording sitesin a
NGI remained to be solved. The most sensitive
area to illumination occupied the posterior half of
either eye. One pair (G4) of the remaining NGIs
running along the cluster responded with graded
depolarizing potentials to illumination on the
anterior half of either eye and with graded
depolarizing and hyperpolarizing potentials to
illumination on the posterior half of the ipsilateral
eye. These results imply that there are complex
connections between the SFs conveying visual
inputs from both eyes and the NGls.
1236 Physiology
CELL-TO-CELL COUPLING BY GAP JUNCTIONS AMONG
RETINAL AMACRINE CELLS OF A CYPRINID.
S. Hidaka, M. Maehara, O. Umino, and Y. Hashimoto. Dept. of
Physiol., Tokyo Wom. Med. Coll.. Shinjuku-ku, Tokyo 162
In the vertebrate retinas, responses to visual stimuli spread
both vertically and laterally. Lateral spread of information is
thought to be mediated by two major cell types, namely
horizontal and amacrine cells. It is widely thought that all types
of horizontal cells are electrically coupled in homologous sets,
whereas, it is unknown whether all anatomical or physiological
subtypes of amacrine cells are coupled or not.
The present study was designed to answerwhat functional
subtypes of amacrine cells are coupled. To address the question,
we have measured tracer-coupling (Vaney, '91) among
physiologically identified amacrine cells using a biotinylated
compound, biocytin (Horikawa and Armstrong, '88), lower
molecular weight (372Da) than the Lucifer yellow (457Da)
which used previously to asses dye-coupling. Based on the
responses evoked by a spot-stimuli (Chino and Hashimoto, ‘86),
amacrine cells (n=90) identified in the retina of dace (Tribolodon
hakonensis) were classified into seven distinct types. All types of
amacrine cells showed extensive tracer-coupling to neighboring
cells of the same cell morphologies by intracellular injection of
biocytin. Patterns of the interconnections between coupled cells
varied among the identified cell types. Localization of the
innterconnected sites was strongly correlated with molphologies
of the cell; soma shape, dendritic field size and stratification in
the inner plexiform layer. Electron microscopic observation of
the innterconnected sites revealed the presence of gap junctions.
These studies demonstrate that homologous sets of amacrine
cells are coupled by gap junctions and multiple clusters of such
homologous sets are present on the whole retinal plane.Cell-to-
cell coupling of homologous sets may occur depending on their
retinal functions.
Lamina-medulla projection in the compound eye in the
cabbage butterfly.
M. Shimohigashi and Y. Tominaga. Biol. Lab., Fac. of
Sci., Fukuoka Univ., Fukuoka.
The compound eye of a cabbage butterfly, Pieris
rarae, has five types of spectral receptors (UV, violet,
blue green and red ones). Violet, blue and green
informations from each ommatidium are relayed to the
lamina neurons by short visual fibers. The long visual
fibers of UV and red receptors run through the lamina
cartridge, terminating to the medulla.
In the present study, we examined the projecting
pattern of neural processes from lamina to medulla by
light and electron microscopies. Axons of lamina
neurons terminated at the distal level in the outer layer of
the medulla. Whilst, the long visual fibers of UV
receptors showed no synaptic profiles in the outer layer
and had lateral branches at the middle level of the
medulla. Six types of medulla neurons were found. Four
of them extended their axons to the lobula complex, but
the two others projected directly to the higher visual
center. On the basis of the level of dendritic processes of
the medulla neurons, it was supposed that spectral
informations relayed by the lamina neurons are
transmitted to the lobula complex via the medulla
neurons, and that spectral information from UV receptor
is sent directly to the posterior slope of the
protocerebrum by the medulla giant neuron.
POSTEMBRYONIC DEVELOPMENT OF THE OPTIC
LOBE OF THE BUTTERFLY: FATE OF THE LARVAL
VISUAL INTERNEURONS.
T. Ichikawa. Department of Biology, Faculty of Science,
Kyushu University, Fukuoka.
The larval optic neuropile of the swallowtail butterfly Papilio
xuthus has two distinct area, lamina and medulla, and is
enclosed by two imaginal disks which are composed of
neuroblasts and develop into an imaginal optic lobe dunng
metamorphosis. Fate of the larval optic neuropile and larval
visual interneurons have been examined by using the reduced
silver impregnation method and the immunohistochemistry of
y-amino butyric acid (GABA). The larval visual interneurons
in the lamina could not be followed during metamorphosis, but
neurons in the medulla could be followed throughout pupal
development because their somata were always significantly
larger than those of other neurons differentiating from the
neuroblasts. The larval medulla neuropile was remained as a
small frontal protrusion of the imaginal medulla. There are
about 100 neurons in the larval medulla. Several neurons were
immunoreactive to the GABA antibody and extended dendntic
processes to the larval lamina and medulla. The medulla
neurons lost those processes at the earliest stage of the pupal
development and differentiated many new processes that
invaded the developing imaginal medulla. The processes
covered a large area of the neuropile. As the neuropiles of the
optic lobe increase in volume, the dendritic processes of the
GABA neurons increased in length and density. Axons of the
neurons run down an optic tract between the labula and the
lobula plate and extended to the lateral area of the proto-
cerebrum. The results demonstrate that at least some larval
visual interneurons are reorganized into imaginal optic lobe
and play a new role in the visual processing of the adult
butterfly.
DISTRIBUTION OF GABA-LIKE IMMUNOREACTIVITY
IN THE BRAIN AND OPTIC NEUROPIL OF INSECTS.
X. Zhao, T. Ichikawa and Y. Toh. Dept. of
Biol., Fac. of Sci., Kyushu Univ., Fukuoka.
Localization of GABA-like immunoreactive
neurons was examined in the brain and
visual system of three species of insects:
adult cockroaches (Periplaneta americana),
swallowtail butterfly larvae (Papilio
xuthus) and tiger beetle larvae (Cicindela
chinensis).
In the cockroach compound eye visual
system, some medulla neurons and some
fibers in the medulla neuropil were GABA-
like immunoreactive. Possible centrifugal
fibers originating in the medulla neuropil
were’stained, but lamina neurons were not
stained. In the dorsal ocellar system it
was reported that possible efferent
neurons might be GABAergic and their
terminals contained dark vesicles together
with clear vesicles. In the present study
GABA-like immunoreactivity occurred in
those efferent neurons. In the larval
visual system of the swallowtail butterfly
and the tiger beetle some medulla neurons
were GABA-like immunoreactive, but no
lamina neurons were immunoreactive. In
both larvae possible efferent fibers were
GABA-like immunoreactive. These data
suggest GABA may play some _ roles in
efferent control as well as in inhibitory
effects of the insect visual system.
Physiology
DESCENDING INTERNEURONS IN THE COCKROACH
PERIPLANETA AMERICANA.
S.Watanabe and Y.Toh. Dept. of Biol., Fac.
of Sci., Kyushu Univ., Fukuoka.
Descending interneurons, which possess
cell bodies in the brain, were examined in
the cockroach by intracellular recording
and staining. Microelectrodes were impaled
at the cervical connective, and responses
of interneurons to several sensory stimuli
were recorded. These interneurons were
classed into nine types on the basis of
effective sensory stimuli. Types 1-3
neurons responded to ocellar illumination
and wind to cerci and antennae. Types 1
and 2 neurons responded to illumination to
ipsilateral and contralateral ocelli,
respectively, whereas type 3 neurons
responded to illumination to either
ocellus. Types 4 and 5 neurons responded
to ocellar illumination only. Type 5
neurons also responded to moving stimuli.
Type 6 neurons responded to compound eye
illumination and wind to cerci and
antennae. Other three types responded to
wind to cerci and/or antennae, but not to
illumination. Of 114 neurons recorded, 108
neurons responded to wind, and 86 neurons
responded to ocellar illumination. These
data suggest that information about wind
detected by antennae and cerci is
integrated together with photic information
detected by ocelli in the brain, and such
integrated information may descend to the
thoracic motor center to modulate
locomotion pattern of the animal.
BLEACHING OF THE DARK BROWN PIGMENT
GRANULES IN THE BUTTERFLY EYE
Y. Shimazaki, E. Eguchi, Dept. of Biol., Yokohama City
Univ., Yokohama
Pigment cells in the compound eye of a butterfly Papilio
xuthus contain dark brown pigment granules. It is said that
these pigment granules seal the light and produce light-
path to retina, so that each ommatidium is optically isolated.
This function of pigment granules is controlled by the
pigment migration according to light and dark adaptations.
Now, we present bleaching of these pigment granules
themselves by light. Firstly, Both light and dark adapted
eyes were fixed, dehydrated and embeded. 4 jm thick
sections through a longitudinal optical axis of the
ommatidia were made. The color of pigment granules in the
light adapted eye was lighter than that in the dark adapted
eye. The color of pigment granules in the dark adapted eye
become pale within 30 min by light irradiation. Secondly,
unfixed eye was frozen in liquid nitrogen, sectioned and
observed with light microscope. The dark brown pigment
granules become pale by light irraduation. This
phenomenon was retained after eye was fixed with 2%
paraformaldhyde and 2% glutaraldehyde for 3 hr. Lastly,
after the removal of the retina from the compound eye,
these pigment granules were isolated from the eye cup by
shaking in Ringer's solution and by sucrose gradient
centrifugation. These pigment granules suspended in 60%
sucrose were used as sample. Spectral absorbances of the
sample were examined under anaerobic condition, before
light was irradiated to the sample and after light was
irradiated to the sample for 30 min. Absorbance (350nm -
800 nm) of post-light irradiated sample was lower than that
of pre-light irradiated sample.
These results indicate a part of dark brown pigment
granules themselves is bleached by light.
1237
EFFERENT CONTROL IN THE ANTERIOR LATERAL
EYES OF ORB WEAVING SPIDERS
S. Yamashita. Biol. Lab., Kyushu Inst.
of Design, Fukuoka.
The light responses of the anterior
lateral(AL) eyes of the orb weaving
spiders, Argiope bruennichii and A.
amoena were controlled by efferent optic
nerve signals transmitted from a
circadian clock in the brain. At night,
the amplitude of ERGs to low light
intensities increased, but those to
intermediate and high light intensities
decreased, i.e. the slope of the
intensity-response curve for the “night
state" was slighter than that for the
"day state". Electrical stimulation of
the optic nerve produced effects on the
ERGs of the AL eye similar to endogenous
efferent activity. Octopamine, a
putative efferent neurotransmitter, also
decreased the amplitude of ERGs to
intermediate and high light intensities.
It was suggested that the effect of
efferent signals on the green spectral
mechanism was greater than that on the
ultraviolet mechanism.
WAVELENGTH DISCRIMINATION IN THE JUVENILE
GOLDFISH.
K. Ohnishi. Dept. of Physiol., Nara
Medi. Univ., Kashihara
Wavelength discriminations of the
juvenile and the adult goldfish were
measured among three spectral sensitivity
maxima (450, 525 and 625 nm) of the
goldfish cones using a go/no-go task.
They were trained to avoid an
electroshock in such a way that they move
from or stay in an illuminated side ac-
cording to the difference in wavelength
of discriminative monochromatic lights.
To prevent the fish from discriminating
the lights in terms of brightness, the
intensity of each monochromatic light was
adjusted to produce equal stimulus ef-
ficiency for discrimination on the basis
of active avoidance learning rate
measured at various light intensities.
Juveniles and adults showed good dis-
crimination ability in 525 nm/625 nm
discrimination, while only juveniles
showed very poor discrimination ability
in 450 nm/525 nm and 450 nm/625 nm
discriminations. In contrast to adults,
very few juveniles trained with the lat-
ter stimuli could acquire discriminative
performances. These results indicate
that the blue-sensitive (or blue-
perceptive) mechanism of juveniles is im-
mature and thus the color vision in
juveniles is different from that in
adults in at least visible spectra.
1238 Physiology
PHOTOPERIODIC RESPONSE IN DROSOPHILA
MELANOGASTER :
EFFECT OF VITAMIN A-DEFICIENCY
A.Okada, I.Shimizu. Center for Ecological Research,
Kyoto University, Kyoto.
Females of the fruit fly,D. Melanogaster ,enter an adult
ovarian diapause under short photoperiods at suitable
temperature(10-13°C),and sensitivity to the diapause
promoting conditions is restricted to the adult stage.It was
reported that carotenoid-deprivation affected the
photoperiodic response in some insect species.We reared
D.melanogaster (Canton-S) on a carotenoid-deficient diet
and a beta-carotene-supplemented diet,and compared the
diapause incidences.The result was that flies on both diets
showed little differences of the diapause incidences at short
photoperiod(8L) between long photoperiod(16L),but that
diapause incidences of the flies on the
beta-carotene-supplemented diet were higher than those of
the carotenoid-deficient flies.
CIRCADIAN RHYTHM IN THE RESPONSIVE-
NESS OF VISUAL INTERNEURONS IN THE
CRICKET OPTIC LOBE
K. Tomioka, M. Ikeda and Y. Chiba. Biol. Inst., Fac.
Sci., Yamaguchi Univ. , Yamaguchi
The longterm record of multiple unit neural activity from the
optic stalk of the cricket Gryllus bimaculatus revealed that the
sensitivity of visual interneurons to 15 min light pulses given
every 2 hr fluctuates in a time of day dependent manner in both
in situ and in vitro preparations. This indicates that the sensitivity
of visual interneurons is under the control of the circadian clock
in the optic lobe. Similar circadian changes can be observed in
single neuronal recordings. We have so far functionally
identified five types of visual interneurons in the medulla. All of
them exhibited a circadian change in their responsiveness. Two
units, which did not fire spontaneously had their responsiveness
to light-on increasing either nocturnally or diurnally. The other
three units had a spontaneous discharge that also changed in a
circadian manner. One of the three units had responsiveness
increasing with the increment of the spontaneous firing
frequency, while in the others the response to light pulses
reversed from excitatory to inhibitory as the spontaneous activity
increased. The results suggest that the circadian clock modulates
not only the sensitivity of interneurons but also the visual
information processing in the optic lobe.
Rhythmicity of chromophore turnover of
visual pigment in the Antarctic amphipod
Qrchomene plebs (Crustacea; Amphipoda).
T.Hariyama , A.Terakita*, and
V.B.Meyer-Rochow?. IResearch Center for
Applied ImEouMaL Lon Sciences, Tohoku
Univ., Sendai, “Biological Inst., Facul-
ty of Edu., Oita Univ., Oita, and
Exp.Zool.& E.M., Univ.of the West
Indies, Kingston, Jamaica
1.Retinal amounts in the compound eye
of the Antarctic amphipod Orchomene
plebs were assessed during conditions of
continuous summer daylight every 3 hours
over a period of 48 hours. The habitat
of the experimental animal is the bottom
of the Ross Sea (78°S; 166°E) down to
depths of at least 400 m; water tempera-
ture is a constant -1.8°C. A periodici-
ty of twelve hours, not of twenty-four
hours, was detected with relative
amounts of ll-cis retinal exhibiting
peaks at midday and at midnight and
troughs at 7:00h and 19:00h. 2.The
result that 90% of retinoid were insolu-
ble in n-hexane suggests that at least
90% of the measured retinoid were at-
tached to membrane-bound proteins such
as opsin. 3.Selective light adaptation
showed that the visual pigments were
thermostable and photoregenerable. The
Main absorbance peak of rhodopsin,
compared with metarhodopsin, seems to be
in the longer wavelengths.
Detachment behavior and enhanced respiration
in adult Japanese lampreys.
R. Kawasaki Biomed.Technol. Niigata Univ.
Niigata, 951 Japan.
Combination SF attachment and detachment
(initiation and elimination of sucker vacuum
is necessary to lamprey feeding, nest-build-
ing and mating.
Anesthetized and spinalized lampreys were
fixed in water at 10 C. The sucker pressure,
nasal respiratory pressures and branchial
EMGs were simultaneously recorded.To initi-
ate detachment behavior, tactile (needles or
bars) or electrical (1V,lms) stimulation was
applied dorsally to the skin of the sucker
funnel. The criterion for the initiation was
the elimination of the pressure-negativity
in the sucker cavity.The sucker was fixed by
pinning it to the plate to avoid detachment,
so the sucker pressure was constantly moni-
tored throughout the experiment.The stimula-
tion initiated enhanced respiration (syn-
chronous augmentation of the EMGs and respi-
ratory nasal pressure) and synchronous clos-
ings (contraction of the constrictor mus-
cles) of all branchial pores,and immediately
followed by the decrease or elimination of
sucker vacuum.The findings indicate that the
enhanced respiration produces rapid movement
of water from the gill pouches into the
sucker cavity to eliminate sucker vacuum.The
rapid detachment to escape from an unpleas-
ant place may occur without involvement of
annular muscles or HS. In conclusion, the
functional significance of so-called "vigor-
ous respiration" may not only be "cleaning
or coughing" but also detachment for escape.
This kind of excitatory activity of the mus-
culatures which surround “branchial intes-
tine" is not only in lamprey's behavior but
also commonly in ‘ejection reflex' in sea
squirts, salps and lancelets.
Physiology 1239
THE ORIENTATION TO FOOD STIMULI BY THE
TENTACLES OF THE PULMONATE SNAIL,
Indoplanorbis exustus.
M.Fujimoto, N.Ueda, M.Hayashi, H.Kunioka and
H.Ohtani. Zool. Inst., Fac. of Sci., Hiroshima Univ.,
Higashi-Hiroshima.
The role of tentacles on the freshwater pulmonate
snail, /ndoplanorbis, for its orientation to food
substances was studied. The animal was perpendicu-
larly fixed in position in the experimental chamber by
holding its shell on a ping-pong ball which can rotate
as the animal creeps on it. This setup allows us to
determine the direction of movement. Each tentacle
was set in the right and left compartment of the
chamber and food substance (lettuce leaves, 1 gr) was
set 3 cm before the animal in either compartment.
About 75 % of the intact animals showed a distinct
turning response. and 85 % of them turned to the
direction of the food. After amputation of either one
or both tentacles, the proportion of the snails that
turned in the correct direction decreased (60-80 %).
The values, however, showed that they can reach food
substance without the aid of tentacles. The application
of lettuce extract on the tip of a tentacle caused a
ipsilateral turning response. A more rapid response
was also observed even from snails without tentacles
when the extract was applied to the basal region of a
tentacle. These results suggest that the tentacles are
involved in food orientation using them for tropotaxis
and klinotaxis. However, the receptor site might be
located elsewhere besides the tentacles.
THE ORIGIN OF AXOTOMY-INDUCED LONG-LASTING
FIRING IN AN IDENTIFIED MOTONEURON OF
THE CRAYFISH.
A. Muramoto. Fukushima Biomed. Inst. of
Environm. & Neopl. Dis., Futaba-gun.
My previous experiments have demonstra—
ted that transection of the abdominal nerve
cord along its long-axis at any level below
the 1st abdominal ganglion (Al) can always
induce a prolonged excitability in an iden-
tifiable motoneuron (AML) of the crayfish.
The origin of this long-lasting firing (LLF)
and its generation site were investigated
in this study. It was found that the LLF
immediately stopped by application of a co-
untercurrent (bucking voltage) to the nerve
cord posterior to the transection site,
while a current (depolarizing pulse) could
induce the LLF in the quiescent state of
AML during the stimulation. Raising the
stimulation strength increased the frequen-
cy durino stimulation-induced LLF in AML.
There was no remarkable difference between
axotomy- and stimulation-induced LLFs.
Depolarizing pulse to the connective above
Al failed to induce a LLF in AML, but this
pulse below Al always could produce such
a response.
It was then concluded that a prolonged
firing of AML following transection must be
due to axotomy of AML itself, in which its
ascending axonal process runs through the
nerve cord, ending to Al, and must result
from a depolarization which formed in its
transection site.
NEUROMUSCULAR TRANSMISSION IN THE RED
MUSCLE OF A PECTORAL FIN OF MUTSUGORO.
T. Hidaka’, K. Kusano“, M. Tanaka“ and Y.
Matsuki2. Dept. of Biol., TPac. of Gen.
Edu. and 2Fac. of Sci., Kumamoto Univ.,
Kumamoto.
Mutsugoro, Boleophthalmus pectinirost-
ris, creeps around using a pectoral fin on
mud flats at low tide but stays in a bur-
row in the mud at high tide. A pectoral
fin muscle of mutsugoro consists of the
red muscle fibers. Some properties of the
neuromuscular transmission were studied by
the electrophysiological methods in this
muscle. The single nerve stimulation elic-
ited three types of junction potential,
excitatory junction potential (ejp), inhi-
bitory junction potential (ijp) and dipha-
sic junction potential (diphasic jp). An
initiation of ejp was more dominant than
ijp and diphasic jp. The action potential
was not initiated, even after the summa-
tion of ejp was produced by the repetitive
nerve stimulation. Three types of junction
potential were augmented by anticholines-
terase, neostigmine, and were suppressed
by nicotinic ACh antagonist, d-TC. A mus-
calinic ACh antagonist, atropine, was less
effective than d-TC. ACh and ACh agonist,
Carbachol,.also decreased all types of jun-
ction potential.
From the results, it was suggested that
the neurotransmitter of this muscle may be
ACh and the nature of the ACh receptor may
be nicotinic.
SPATIAL DISTRIBUTION AND ELECTROPHYSIOLOGI-
CAL CHARACTERISTICS OF NEURONS ASSOCIATING
WITH SPERMATOPHORE-FORMATION IN MALE CRICK-
ET, TELEOGRYLLUS COMMODUS .
A.Nakanishi and N.Ai. Dep.of Biol.,
Tokyo Gakugei Uni., Koganei, Tokyo.
In adult male crickets,their cercal muscles
(M1) ,muscles linked to reproductive organs
(accessary glands, epididymis and ejacula-
tory duct) (M2) and phallic muscles (M3) are
respectively innervated by N9-1,2 and 3
branch from the 9th lateral root (N9) in
the terminal abdominal ganglion (AG-V). Each
branch has bilaterally about 6(N9-1) or 10
(N9-3) somata of motor-neuron in the postero-
lateral region of AG-V. But in N9-2, 130 or
more somata are appeared along the midline
of AG-V with the bifurcating processes ex-
tend symmetrically to both sides. Consider-
ing from results of vital and back-fill
staining,these neurons would be DUM ones for
M2 muscle group. However,only 4 to 6 somata
of them have been stained with their axon
processes extended to only one side of the
root looks like motor-neuron.
By electrical activity in N9 branch, effer-
ent spikes are recorded with higher freq-—
uency and different amplitude in N9-2 than
the others. And by electrical responses are
appeared as more long lasting contraction in
M3 (N9-3) than in M1 (N9-1) -. N9-2 root has in-
nervation to muscle group with automatic and
continuous contraction and N9-1 root with
temporaly contraction for cercal muscle.
1240 Physiology
RADULAR MECHANOSENSORY NEURON IN _ THE
BUCCAL GANGLIA OF THE, TERRESTRIAL SLUG.
.Kawahara’, M.Yano2, and H.Shimizu’.
Fac. of Paamuecettleall Sei, Uniiven ot
Tokyo, Tokyo, Res. Inst. of Electrical
Communication, Tohoku Univ., Sendai.
A radular mechanosensory neuron, RM, was
identified in the terrestrial slug, Inci-
laria fruhstorferi. A pair of RMs were
located symmetrically on the posterior
edge of the dorsal surface of the buccal
ganglia. Fine neurites ramified within the
buccal ganglia. Main neurites exited from
buccal nerve 3s (n3) and entered the
subradular epithelium. When the posterior
part of radula was distorted by bending,
afferent spikes of RM, which were preceded
by the axonic spikes recorded at n3, were
observed. his response remained after
removing Ca from saline. RM was suggest-—
ed to be a primary mechanoreceptor. When
rhythmic buccal motor activity was evoked
by food application or nerve stimulation,
RM received excitatory input in the rasp
phase, in which the supramedian radular
tensor (smt) was active. Afferent spikes
of RM, evoked by electrical stimulation to
the radula, broadened synchronously with
this phasic input. Spike broadening of RM
resulted in an increase of the amplitude
of excitatory postsynaptic potential in a
motoneuron which projected to smt. These
results suggested a phase-dependent senso-
ry modulation in the feeding system of the
slug.
DISTRIBUTION OF CATECHOLAMINE-CONTAINING
NEURONS IN THE CEREBRAL GANGLIA OF THE SLUG,
INCILARIA FRUHSTORFERI.
Y.Makino!, S.Kawaharal!, M.Yano2, H.Shimizu!. 1Fac. of
Pharmaceutical Sci., Univ. of Tokyo, Tokyo, 2Res. Inst.
of Electrical Communication, Tohoku Univ., Sendai.
The distribution of catecholamine-containing (CA)
neurons in the cerebral ganglia of the slug Incilaria
fruhstorferi was studied using the formaldehyde-
glutaraldehyde and glyoxylic acid histofluorescence
methods. Approximately 200 CA neurons were observed
in each hemiganglion. Most of CA neurons had small
cell bodies (5-20 j1m in diameter), which were
aggregated into five discrete clusters. Several larger CA
neurons (30-50 tm in diameter) were scattered mainly
on the dorsal surface. CA nerve fibers were observed in
the neuropile region, cerebral commissure, and all
cerebral nerve roots. In the somatic region of the
procerebrum (PC), there were extrinsic CA _ fibers,
which had distinctive varicosities. The extrinsic fibers
entering the PC were derived from two thick bundles of
CA fibers. One bundle was on the dorsal surface of the
neuropile of the PC, and continued to tentacular nerve.
This bundle consisted of the CA fibers from the cluster
of small neurons located just around the medial edge of
metacerebral giant cell and two dorsal large CA
neurons. The other bundle was in the neuropile of
anterio-ventral region of the cerebral ganglion. This
bundle consisted of fibers from the ipsi- and contra-
lateral clusters of small CA neurons located in close
vicinity to the PC
DISTRIBUTION OF SEROTONIN- AND FMRFAMIDE-
LIKE IMMUNOREACTIVITY IN THE BRAIN OF SILK
MOTH BOMBYX MORI.
A.Iwamal, A.Nakagawa! and R.Kanzaki2.
1Tsukuba Research Center, SANYO Electric
Co., Ltd., Tsukuba and 2Inst.of Biol.Sci.,
Univ. of Tsukuba, Tsukuba.
Distribution of serotonin- and
FMRFamide-like substances was respectively
studied in the brain of silk moth Bombyx
mori using immunohistochemical technique.
Approximately 180 Serotonin-like
immunoreactive(SLI) somata were found in
the brain. Most of them were located in
the optic lobe and median protocerebrum
and some of them were found in the
deutocerebrum and suboesophageal ganglion.
FMRFamide-like immunoreactivity (FLI) was
detected in more than 800 somata in the
brain. These were found in the optic lobe,
median protocerebrum, deutocerebrum and
suboesophageal ganglion. Both SLI and FLI
fibers were distributed extensively
throughout the neuropil areas, especially,
the optic lobes, antennal lobes, mushroom
bodies, lateral accessory lobes, and
central body. It was reported that the
antennal lobes, mushroom bodies and
lateral accessory lobes were included in
the pathway of the pheromonal information
processing. These results suggest that
serotonin and FMRFamide might serve
several functions as neurotransmitters or
neuromodulators in the nervous system
related to the mating behaviour.
NEURONAL ARCHITECTURE OF THE MUSHROOM BODY
IN THE HONEYBEE BRAIN AS REVEALED BY FMRF-
AMIDE AND SEROTONIN IMMUNOHISTOCHEMISTRY.
A.Iwama!, A.Nakagawa!, A.Mizukamil AND T.
Shibuya2. !1Tsukuba Reseach Center, SANYO
Electric Co., Ltd., Tsukuba and ?Inst. of
Biol. Sci., Univ. of Tsukuba, Tsukuba.
The mushroom bodies are prominent
structures in the honeybee brain. To know
their functional organization, we have
studied the distribution of FMRFamide- and
serotonin-like substances in the mushroom
bodies of the honeybee brain using immuno-
histochemical technique.
Each mushroom body neuropil consists of
two calyces connected by two stalks to two
lobes. These neuropil regions contain two
types of neuronal elements, the intrinsic
and the extrinsic fibers. The intrinsic
fibers are stratified across the stalks
and lobes, based on their cell types.
Serotonin-like immunoreactivitiy (SLI) was
found at widespread extrinsic fibers in
the lobes and stalks. While FMRFamide-like
immuno-reactivitiy(FLI) was found in some
layers of the lobes and stalks, equivalent
to intrinsic fiber bundles. The somata and
the dendrites in the calyces of intrinsic
cells did not show FLI in the adult brain.
However, in the pupal brain, the calyces
and the somata located at the outersurface
of the calycal neuropil showed FLI. The
results represent the basic architecture
of the mushroom bodies and suggest that
FMRFamide and serotonin may serve
important roles in the mushroom bodies.
Physiology 1241
DEVELOPMENT OF THE CNS AND SEROTONIN-
IMMUNOREACTIVE PROCESSES IN THE
OPISTHOBRANCH MOLLUSC PLEUROBRANCHAEA
JAPONICA.
K. Ohsuga and K. Kuwasawa. Dept. of Biol.,
Tokyo Metropolitan Univ., Tokyo.
We studied development of the CNS an
distribution of serotonin-immunoreactive
neurons during a period from the embryo to
the young juvenile just after metamorphosis
in Pleurobranchaea japonica.
We observed the CNS with thin sections
obtained from preparations embedded in
Quetol-812 under a light optics. The basic
shape in the CNS such as the cerebro-
pleural, pedal and buccal ganglia and the
rudiments of the rhinophore and oral veil
ganglia was settled during the veliger
phase. The rhinophore and oral veil
ganglia were extruded from the cerebro-
pleural ganglion after metamorphosis.
Processes serotonin-immunoreactive to
anti-serotonin antiserum were found in
thick paraffin sections and whole mount
preparations by means of PAP and FITC
method for, respectively, paraffin sections
and whole mounts. At the veliger stage
serotonin-immunoreactive cells were
observed in the cerebro-pleural and pedal
ganglia. A cluster of immunoreactive cells
was found on the larval cerebral
commissure. The cluster sent processes
along edges of paired velar lobes. After
metamorphosis the cluster on the cerebral
commissure disappeared. It is likely that
the cluster was a specific neural
organization for the veliger.
RESPONSE OF OSCILLATORY FIELD POTENTIAL TO SOME
CONDITIONED ODORS IN SLUG'S BRAIN.
T.Kimura, H.Suzuki, A. Yamada, T.Sekiguchi and
A.Mizukami. Tsukuba research center, SANYO ELECTRIC
Co. LTD., Tsukuba.
To understand the mechanisms of olfactory
recognition and leaning, we analyzed odor-
information flow and its expression in the slug's
brain using morphological and physiological
technique.
An odor information caught by numerous sensory
cells on tentacle tip was transferred to input mass
of pro-cerebral lobe and meso-cerabral robe of brain
via tentacle ganglion. The pro-cerebral lobe (PCL)
was divided morphologically into three mass (cell
mass, input mass and output mass). The cell mass was
a cluster of cell body of intrinsic neurons, of
which process elongated into output mass passing
through input layer.
Local field potential (LFP) was recorded from the
cell mass or output mass of PCL on tentacle-brain
preparation which dissected from the body. When the
appetitive-conditioned odor was applied on the
tentacle tip, the frequency of LFP increased during
Stimulation. However, application of aversive-
conditioned odor decreased the frequency. Similar
results were obtained on the output-mass of PCL-
tentacle preparation which was dissected from meso-
cerebral lobe.
These observations suggested that an olfactory
stimulus sensed on the tentacle tip was transferred
into PCL and recalled the memory associating with
the odor in it.
PROPERTIES OF DUAL EXCITATORY INNERVATION OF
THE UROPOD MUSCLES IN CRAYFISH.
T. Higuchi. Dept. Gen. Edu., Higashi-Nippon-Gakuen Univ. ,
Ishikan-Tobetsu, Hokkaido.
In crayfish uropod, neuromuscular responses in muscle fibers
of both tonic and phasic muscles which received dual excitatory
innervation were observed in order to know the functional
relationship between the two excitatory motor neurons. All of the
muscle fibers investigated in such muscles exhibited no
characteristic difference in their innervation pattern and in their
neuromuscular responses. This suggested that the two excitatory
motor neurons within the muscles equally distributed their
terminals to all muscle fibers. In tonic muscle fibers, it appeared
that two simultaneously observed trains of excitatory junctional
potentials, which accurately reflected the activity of each
excitatory motor neurons, were independent from each other,
both in the absence and the presence of stimulations. The
phenomena showing the direct or close interaction between the
two excitatory motor neurons were not observed either centrally
or peripherally except for the simple summation of the excitatory
junctional potentials induced by the activity of individual excit-
atory motor neurons. In phasic muscle fibers, whose excitatory
motor neurons were normally silent, no peripheral interaction
between the two excitatory motor neurons was also suggested by
the observation of excitatory junctional potentials and active
responses induced by selective electrical stimulation of individual
neurons. Central interaction, however, is unknown. An interest-
ing feature was the different action of the individual excitatory
motor neuron which innervated the adductor exopodite.
Postsynaptic events induced by one of the two excitatory motor
neurons was apparently rapid fatigue, while the action of another
was constant. The meaning of this difference needs to be
resolved. It is likely that individual excitatory motor neurons act
independently on demands of different behavioral performances.
NONLINEAR ANALYSIS OF CERCAL SENSORY
PATHWAY
T.Shimozawa, Y.Baba and T.Shimizu. Lab.
of Neuro-Cybernetics, Res. Inst. for
Electronic Sci., Hokkaido Univ. Sapporo.
Signal transmission pathway to giant
interneuron 8-1 in the cricket cercal
sensory system was clarified by using
Wiener's white noise analysis. Air
current stimulus was modulated with a
Gaussian white noise signal of 500 Hz
band width. Responses of the interneuron
were recorded by intracellular electrode.
The stimulus and response waveforms were
stored on a digital-audio-tape recorder
and collected into a workstation through
GPIB interface. The linear signal
transmission was estimated from the
cross-correlation between stimulus and
response. The 2nd order cross-correlation
between stimulus white noise at two
different times and response indicates
the contribution of 2nd order
nonlinearity to the signal transmission.
Nonlinear signal transmission gave a good
clue to determine the sequence of
processing element in signal flow to the
interneuron. Interneuron 8-1 was revealed
to receive bilateral inputs. Both showed
strong amplitude saturation but with
different threshold. The high threshold
input has 1 ms delay and was subtracted
from the other at the interneuron.
Subtraction of delayed signal helps to
detect the rate of change of stimulus.
1242 Physiology
Input pathway of cricket cercal nonspiking
interneuron revealed by Wiener's white
noise analysis. Y. Baba, T. Shimizu and
T. Shimozawa Lab. of Neuro-Cybernetics.
Res. Inst. for Electronic. Sci., Hokkaido
Univ. Sapporo.
Cercal sensory nonspiking interneuron,
LNI-7 was studied by using Wiener's white
noise analysis. Cerci were stimulated by
an air current modulated with a Gaussian
white noise. Band width of the white
noise was 500 Hz. The first- and second-
order Wiener kernels were extracted by
cross-correlation between the input white
noise and neural response. The extracted
kernels predicted the actual neural
response with mean square error of 58% in
1st order model and of 50% in 2nd order
model. The first-order kernel is tri-
phasic waveform with 6msec latency. The
second-order kernel had elongated nine
eyes which indicates a neural smoothing
after a nonlinior element. At strong
stimulus, small nine eyes before the
elongated nine eyes appeared. When cercus
contralateral to the soma was covered, the
small nine eyes disappeared, whereas the
first-order kernel hardly changed. The
data suggest that LNI-7 has a direct,
inverse high threshold small amplitude
input from the contralateral cercus.
CENTRAL PROJECTION OF THE COLD RECEPTOR
AXON ONTO THE DEUTOCEREBRUM OF THE
COCKROACH.
M. Nishikawa, F. Yokohari and T. Ishibashi. Biol. Lab.,
Fac. of Sci., Fukuoka Univ., Fukuoka
There are two types of thermoreceptive sensilla
on the antenna of Periplaneta americana. One type of
the sensilla contains four receptor cells: a cold, a dry ,a
moist and an unknown cell. The other has a cold
receptor and two or three olfactory receptors.
The central projections of these receptors were
examined by extracellular staining of these axons and
by reconstruction of the plastic sections. One of the
axons from each sensillum, which is in proximal part of
the flagellum, projects into the ordinary glomerulus in
the ventro-posterior and marginal region of the antennal
lobe. These axons are suggested to belong to the cold
receptors. The putative hygroreceptor axons project into
the ventro-posterior glomeruli closely located to the cold
receptor axon terminal. Two or three axons, projecting
to the ventro-medial glomeruli, are suggested to belong
to the olfactory receptor cells.
Cold-sensitive output interneuron, which was
excited by the cold air stimulation on cold receptors in
the medial part of an antenna, innervated a single
glomerulus in the ventro-central region of the antennal
lobe. This glomerulus is different in its location from the
glomerulus where the cold receptor axon terminated as
mentioned above. From these observations we
assumed that the cold receptor axons project into
several different glomeruli corresponding to the location
of the cold receptors on the antennae.
EFFECTS OF NEUROTRANSMITTERS ON DUM NEU-
RONS IN THE COCKROACH THORACIC GANGLIA.
H. Washio. Lab. Neurophysiol. Mitsubishi
Kasei Inst. Life Sci. Tokyo
Effects of putative neurotransmit-
ters, L-glutamic acid, gamma aminobutylic
acid (GABA) and acetylcholine (ACh), on
the excitability of dorsal unpaired median
(DUM) neurons in the thoracic ganglia of
the cockroach, Periplaneta americana, were
investigated using intracellular recording
technique. Bath application of these
neurotransmitters modified the excitabili-
ty of the DUM neurons associated with the
membrane conductance change. L-glutamic
acid caused a strong, but ACh had a week
desensitization of the firing activity,
while GABA had an inhibitory action on the
excitability. No appreciable changes in
Membrane potential were elicited with bath
application of these neurotransmitters at
the concentration of 107°M, in spite of
modification of the excitability. However
10-4M L-glutamic acid caused a transient
depolarization and 10-4M GABA caused a
long-lasting depolarization. The results
are compatible with central nervous synap-
tic transmission onto thoracic DUM neurons
being mediated by L-glutamic acid, GABA
and ACh.
DIFFERENTIATION OF DUM NEURONS ON THE
LAST ABDOMINAL GANGLION IN FEMALE
CRICKET, TJTELEOGRYLLUS COMMODUS.
T. Ai and N.Ai. Dept.of Biol.
Tokyo Gakugei Univ., Koganei, Tokyo.
We have already presented that five
clusters of DUM neuron in adult female
cricket are oriented on the dorsal
surface of the last abdominal ganglion
(AG-\). The neurons of four clusters
(C-1, 2,3 and 4) innervate to both the
common oviduct and the lateral oviduct
and the other neurons located in the
fifth cluster (C-5) innervate to the
tergal muscle (M-4), the intervalvular
muscle (M-5,6), the targosternal muscle
(M-7) and the common oviduct. In adult
stage, these DUM neurons are stained by
neutral red which is well known to stain
monoaminergic neuron specifically.
However, in the last larval inster
(LLI) stage, all of five cluster on DUM
neuron in AG-V) fully innervate as the
same manner as that of adult, and so
the DUM neuron of C-1,2 and 3 in LLI
stage are stained by neutral red, but
these of C-4 and C-5 are not clearly
stained in some of them.
Spontaneous activity of DUM neurons in
LLI stage are enough recorded as same as
that in adult preparation
In other hand, we observed on preparat-
ion in LLI stage that M7 fiber has been
fully observed its striation and evoked
contraction yield by electrical stimula-
tion to root N7-1, but MS fiber not
clearly observed its striation and
evoked one also not yield
Physiology 1243
MORPHOLOGY AND PHYSIOLOGICAL PROP-
ERTIES OF DORSAL UNPAIRED MEDIAN (DUM)
NEURONS IN THE CRICKET TERMINAL
ABDOMINAL GANGLION.
B. Chen and T. Yamaguchi. Dept. of Biol., Fac. of
Sci., Okayama Univ., Okayama.
Numerous DUM neurons are located in several
clusters in the terminal abdominal ganglion (TAG)
of the cricket. The morphology and physiological
properties of individual DUM neurons in each
cluster were studied by intracellular recording and
dye injection techniques to reveal their functional
roles in the sensory information processing or
motor pattern generation in the TAG. So far as we
have examined, various DUM neurons was
divided into the type-1 and type-2 neurons. Most
of the DUM neurons were the type-1 neurons,
each of which bifurcated a pair of axons running
through one of the nerve roots in each side and
innervating the visceral muscles. This type of DUM
neurons showed spontaneous impulse discharges
with a high frequency, but scarcely responded to
wind stimulation of the cerci and to electrical
stimulation of the ventral nerve cord. The
remaining neurons were the type-2 neurons, each
of which bifurcated two or three pairs of axons
running through two or three nerve roots in each
side and innervating the skeletal muscles. These
of DUM neurons responded to wind stimulation as
well to electrical stimulation.
Stretch Receptor Organs in the Thorax and Abdomen
of Ligia exotica (Crustacea, Isopoda)
Y. Takatsuki, A. Niida and T. Yamaguchi. Dept.of
Biol., Fac. of Sci., Okayama Univ., Okayama
Two types of stretch receptor organs(SRO) were
bilaterally found through the thorax to the abdomen
except for the 2nd thoracic and 6th abdominal seg-
ment. Such an SRO comprised a single receptor
muscle and two morphologically distinct sensory cells,
each having a club-shaped dendrite or three-forked
dendrites. The sensory cells with three-forked den-
drites through all segments showed a slowly adapting
response exclusively to stretch stimulus, while the
sensory cells with a club-shaped dendrite(C-type sen-
sory cell) in the abdomen responded with rapid adap-
tation irrespective of degrees of stretch amplitude.
Similarly the C-type sensory cells of the thorax
exhibited a rapidly adapting response within the limits
of physiological stimulation. However, a little larger
degree than those of physiological stimulation
produced grouping discharges in the C-type sensory
cells throughout a maintained stretch. Concomitant
impulse frequency of the grouping discharges
increased with stretch amplitude. The same could be
caused by injection of depolarizing current into the C-
type sensory cells. These characteristics might be
attributable to a transitional form between a slowly
and rapidly adapting SRO in this animal.
Dynamics of a Negative Feedback Loop
Underlying Resistance Reflex of Leg Motor
Neurones in the Locust
Y. Kondoh! and P. L. Newland?, ‘Honda R&D Co. Ltd., Wako
Research Center, Saitama and @Department of Zoology, Uni-
versity of Cambridge, Cambridge, U.K.
Imposed movements of the femoral-tibial joint of the locust
metathoracic leg elicit a resistance reflex in the extensor and
flexor motor neurones of the tibia. A femoral chordotonal organ
(FCO) encodes movements about the femoral-tibial joint and
provides feedback to the leg motor neurones that acts to main-
tain a stable posture of the leg. We have analyzed the response
dynamics of individual neuronal elements involved in this local
reflex pathway (or feedback loop) by means of Wiener kernel
methods.
Intracellular recordings were made from FCO afferents and
extensor and flexor motor neurones of the tibia while moving
the FCO apodeme with a band-limited Gaussian white noise
signal. First- and 2nd—-order kernels were computed by a
cross-—correlation between the response and the white noise to
define the input-output characteristics of the neurones. First-
order kernels of the FCO afferents were of 6 basic types that
encode position, velocity and acceleration in 2 directions. The
1st-order kernels of fast and slow extensor motor neurones
were both of a low-pass type having an initial hyperpolarization
followed by a small depolarization, whereas those of some slow
flexors were also low-pass but had an initial depolarization. The
1st-order kernels of the fast and intermediate flexor motor
neurones were differential, and initially depolarizing. Thus, the
extensor and slow flexor motor neurones receive input from
position sensitive FCO afferents, but with opposite directional
responses, whereas the fast and intermediate flexors receive
input from velocity sensitive afferents.
PATTERN OF AXON GROWTH OF MECHANOSENSORY
NEURONS OF MICROCHAETES ON THE DROSOPHILA
NOTUM
A. Usui, K.-l. Kimura. Laboratory of Biology,
Hokkaido University of Education, Iwamizawa
Campus, lwamizawa, Hokkaido
We have followed the axon growth of
microchaetes on the notum, using monoclonal
antibody (Mab) 22C10 which labels neurons and
accessory cells. The spatial pattern of neuronal
pathways is highly reproducible (Fig.1). At 18 hr
after puparium formation (APF), neurons of all
microchaetes are recognized and they begin to send
out axons at 20 hr-APF. At the beginning of
Outgrowing, axons grow toward the particular
direction. The elongating axon contacts and
fasciculates with the
axons of the nearest
neighboring microchaetes.
By 28 hr APF, all axons
finish fasciculation among
them, and they then follow
the axons of macrochaetes
and pre-existing cells,
which continue to be
labeled with Mab 22C10.
The axon of microchaetes
may easily reach distant
target by following the
axons of macrochaetes and
pre-existing cells.
1244 Physiology
MORPHOLOGY AND REGENERATION OF TENTACLE
GANGLION IN SLUG, LIMAX FLAVUS.
H. Suzuki, T. Kimura, A. Iwama, and A. Mizukami.
Tsukuba Research Center, Sanyo Electric Co.
LTD., Tsukuba.
In this study we examined the morphological
aspect of the regenerating posterior tentacle
ganglion of the terrestrial slug, Limax flavus,
to investigate the mechanism of reconstruction
of neuronal networks in the ganglion. Its neural
structure was studied by backfilling the tentacle
nerve with hexamminecobalt chloride. The normal
ganglion included 5 types of neurons. Four types
of them projected axonal processes to the brain.
Two types of the projected neurons shown FMRFamide
immunoreactivity.
The tentacle regeneration usually completed
within about 1 month. That process was divided
into 5 stages. At stage Ill renewal neurons elongated
their neurites into the tentacle nerve. They had
also FMRFamide immunoreactivity. These characteris-—
tics were not observed at stage II. The present
results suggest that the significant differentia-
tions of the regenerated cells begin on stage Il.
MORPHOLOGICAL CHANGES OF THE LATERAL LINE
SYSTEM DURING T4—INDUCED METAMORPHOSIS IN
THE NEOTENIC AXOLOTL, AMBYSTOMA MEXICANUM.
H.-A.Takeuchi!, H.Uehara!, K.Yokota! and
T.Nagai2 ‘4Dept. of Biol., Fac. of Sci.,
Shizuoka Univ., Shizuoka and “Dept. of
PiMvsioil, 5 Weenie yoy Wai, Sela. Ox Mach s 5
Tokyo.
The lateral line system functions as
mechanosensor sensitive to small water
displacement in many aquatic vertebrates,
as well as electrosensor sensitive to weak
electric field in some species. The
degree of metamorphic change of the later—
al line system is very different in the
orders of the class amphibia: most anurans
completely lose their sensory organs and
nerves during metamorphosis, whereas most
ulodeles show little changes. In the
present study, we investigated morphologi-
cal changes of the lateral line system
during thyroxine(T4)-induced metamorphosis
in the neotenic axolotl.
T4 immersion (1.29x10-7M T4) or injec-
tion (1.0nmol T4/gBW) completely metamor-
phosed axolotls in 21-88 days. Their
mechanosensory and electrosensory organs
intensively regressed and disappeared
around metamorphosis, whereas their later-
al line nerves were retained. In the
brainstem, transganglionically transported
cobaltic lysine demonstrated the presence
of the mechanosensory and electrosensory
afferent fibers and the efferent neurons
of the lateral line system at least 3.5
months after metamorphosis.
MOTOR PATTERN GENERATION OF THE POSTERIOR CARDIAC
PLATE _PYLORIC SYSTEM IN THE STOMATOGASTRIC GANGLION
OF THE MANTIS SHRIMP SQUILLA ORATORIA.
K. Tazaki. Biol. Lab., Nara Univ. Educ., Nara.
Activity patterns of the constituent neurons of
the posterior cardiac plate (pcp)-pyloric system in
the stomatogastric ganglion of the mantis shrimp
Squilla oratoria were studied by recording sponta-
neous burst discharges intracellularly from neuron
somata. These neurons were identified electrophysi-
ologically, and synaptic connections among them
were qualitatively analyzed. The pcp constrictor
(PCP), pyloric constrictor (PY), pyloric dilator
(PD), and ventricular dilator (VD) motoneurons, and
the pyloric interneuron (AB) were involved in the
Ppcp-pyloric system. They generated sequentially
patterned outputs. Most commonly, the PCP neuron
activity was followed by the activity of the PY
neurons, and then by the activity of PD/AB neurons,
and VD neuron. The motoneurons and interneuron were
connected to each other either by electrical or by
inhibitory chemical synapses, and thus constructed
a characteristic neuronal circuit which was struc-
turally similar to the homologous pyloric circuit
of decapods. The network properties of the pcp-
pyloric system in stomatopods were phylogenetically
compared to those of the pyloric system in decapods.
All the cell types could produce slow burst-forming
potentials which led to repetitive spike discharges.
Homologous cell types could be observed, but the
number of neurons was variable. The main differ-
ence between the two circuits was the synaptic
connectivity among motoneurons: there were more
electrical synapses in stomatopods, whereas more
inhibitory synapses were found in decapods.
AN OLFACTORY SYSTEM SPECIFIC PROTEIN IN
SOCKEYE SALMON AND KOKANEE SALMON.
M.Shimizul-2, H.Kudol-3, H.Uedal, A.Hara#,
K.Shimazaki2 and K.Yamauchi3. 1lToya Lake
Stn. for Environ. Biol., Abuta-gun, ?Res.
Inst. of North Pacif. Fish., Hakodate,
3Dept. Biol., Hakodate, *Nanae Fish Cult.
Exp. Stn., Kameda-gun, Fac. of Fish.,
Hokkaido Univ.
We have begun to investigate actual
functions of olfactory organs in the
course of imprinting and homing behaviors
in salmonid. Proteins restricted to the
olfactory system (olfactory epithelium,
olfactory nerve and olfactory bulb) and to
the telencephalon of sockeye salmon and
kokanee salmon (Oncorhynchus nerka) were
compared by sodium dodecyl sulfate
polyacrylamide gel electrophoresis in the
presence of 2-mercaptoethanol. Three
protein bands of molecular weight 20, 24
and 40KDa were observed only in the
olfactory system. A polyclonal antibody
to a 24KDa olfactory protein was raised in
a rabbit. The specificity of the anti-
serum was examined by immunoblotting; the
antiserum recognized only one 24KDa band
in the olfactory system, which was absent
from the telencephalon. Similar olfactory
specific proteins of molecular weight
24KDa were recognized in both chum salmon
(Q. kKeta) and masu salmon (Q. masou).
Thus, all salmonids examined to date
possess a 24KDa protein specific to the
olfactory system.
Physiology 1245
DIFFERENTIAL ODOR RESPONSIVENESS ON
REEVE'S TURTLE ACCESSORY OLFACTORY BULB
T. Hatanaka, Dept. of Biol., Fac. of
Educ., Univ. of Chiba, Chiba.
One hypothesis for coding of odor
quality in vertebrates is that receptor
neurons selectively sensitive to partic-
ular odor properties are topographically
segregated in the olfactory epithelium.
Some evidences of topographical ordering
in the olfactory bulb have been demon-
strated in several species. In order to
ascertain that the same olfactory discrim-
inatory process was functioning in the
accessory olfactory system, Reeve's
turtles with developed vomeronasal system
were studied. Induced wave responses. to
several odorants were recorded from five
points of accessory olfactory bulb (ante-
rior, posterior, lateral, medial and
intermediate portion of the AOB), and each
response amplitude was compared.
Responses at anterior portion of the
AOB tended to be larger than those at
other portions when iso-amyl acetate odor
was applied. While, large responses were
recorded at posterior portion to formic
acid vapor and ammonium chloride solution.
Weak responses to propionic acid odor had
no tendency. So, possibility of spatial
coding of odor quality on the turtle
accessory olfactory system was also shown.
PROPERTIES OF IP3-ACTIVATED ION CHANNEL IN
FROG OLFACTORY RECEPTOR CELL MEMBRANE
N. Suzuki
Zool. Inst., Fac. of Sci., Hokkaido Univ. Sapporo
To elucidate the role of IP3-activated ion channels in
vertebrate olfactory transduction, I have studied the properties
of IP3-activated ion channels in inside-out membrane patches
excised from olfactory vesicle and soma membrane of frog
olfactory receptor cells. In a microflow superfusion chamber,
patches were perfused with low calcium intemal KCl-solution
or KF-solution (Ca2+: 1x 10-8 M), in which 1 - 5.3 uM IP3
was dissolved. With Ba-external solution ( Ba2*+: 88 mM ),
flickering channel openings were observed at luM IP3. The
response latency varied from 24 to 1400 msec in different
patches. The shortest latency, however, was comparable to
that for cAMP-activated channel response to 1uM cAMP. The
event histogram analysis revealed that the open time of the
channels became longer in higher stimulus concentrations of
IP3. The channel activity exhibited the adaptive nature and
disappeared within SO - 60 sec when stimulated by 5.3 uM
IP3. The unit conductance at - 85 mV(corrected for the offset
potential) was about 20 pS. The analysis of success rate for
recording of single channel activity in different patches from
different membrane loci suggested that the channel density for
IP3-activated channels was higher in olfactory vesicle
membrane than in soma membrane, but was much lower than
that for cAMP-activated channels.
MEMBRANE CONDUCTANCES RELATED WITH DIVA-
LENT CATIONS OF THE OLFACTORY RECEPTOR
CILIA
T. Nakamura, T. Sato and S. Miyamoto. De-
partment of Applied Physics and Chemistry,
The University of Electro-Communications,
1-5-1 Chofugaoka, Chofu-shi, Tokyo 182
We used the "ciliary patch" configura-—
tion according to Kleen and Gesteland
(1991a) to examine the total membrane con-
ductance of the olfactory receptor cilium
of the bull frog, and recorded the I-V
curves during the application of various
chemicals into the intracellular side of
the cilium and integrated more than 8
curves to detect a small conductance
change. First, we observed the chloride
conductance gated by Canin as reported by
Kleen and Gesteland (1991b), which was,
however, not gated by Mg Then we used
the chloride free solution to examine the
blocking effect of divalent cations on the
cyclic nucleotide gated channel. We found
that the blocking by Ca‘t* is rather weak
compared to that by Mgt. On the other
hand, we could not observe the conductance
change when we applied IP3 up to 50 uM
into the cilium, which suggests that the
electrical change induced by IP3 in the
whole cell configuration reported by other
group might not be due to the Ca influx
Gin wey Cililsie. The overall contributions
of these divalent cations to the membrane
conductance in the cilia are still to be
studied.
CROSS-ADAPTED RESPONSES IN THE MOUSE TASTE
CELL.
K. TONOSAKI. Dept. of Oral Physiol., Sch.
of Dentistry, Asahi Univ., Gifu.
Taste stimulus adsorption is believed
to occur at the taste cell microvillous
membrane. Little is known about the
mechanisms of taste transduction, due to
the technical difficulties of inserting a
glass micro electrode into the mammalian
taste cell. I previously reported that
the mouse taste cell response to a_ sucrose
stimulus is a membrane depolarization
accompanied by an increase in membrane
resistance. The sucrose response increases
in amplitude as the membrane is depolarized
and decreases in amplitude as the membrane
is hyperpolarized. The same taste cell
responds to NaCl with an induced
depolarization of the membrane potential
accompanied by a decrease in membrane
resistance. The NaCl response increases in
amplitude as the membrane is hyperpolarized
and decreases in amplitude as the membrane
is depolarized. These results suggest that
sucrose and NaCl have quite different
response generation mechanisms.
I now present evidence that sucrose pre-
adaptation suppressed the cross-adaptation
responses to NaCl. Intracellular
recordings from receptor cells are
appropriate to understanding cellular
adaptation properties.
1246 Physiology
‘YHE EFFECT OF CYCLIC NUCLEOTIDES ON HE
LABELLAR SALT RECEPTOR AND IlS ADAPTATION
IN THE FLESHFLY.
M. Koganezawa and I. Shimada.
Dept. Biol. Sci., Tohoku Univ., Sendai.
The common role of cyclic nucleotides
has now become noticed in the molecular
mechanism of sensory transduction. Amakawa
et al. (1990) found a stimulating effect of
dibutyl cyclic GMP (dbcGMP) on the labellar
sugar receptor of the blowfly. Why did the
effect restrict to the sugar receptor?
We tested the effect of several nucleo-
tides on the labellar taste receptors of
the fleshfly and analyzed the adaptation
process. One hundred mM dbcGMP was found
to be the most effective on the salt re-
ceptor, while other nucleotides such as cGMP,
8bcGMP and 5'-GMP were less effective.
Adaptation curve of dbcGMP was obtained by
adding that of adaptive cGMP and non-adap-
tive 8bcGMP. Dibutyl cGMP may stimulate
the salt receptor not only by binding to a
nucleotide receptor site but also by per-
meating the receptor membrane. The:tendency
was Similar to the result of the sugar re-
ceptor by Amakawa et al., but it was on the
salt receptor in our experiment. These
findings may suggest a common role as a
second messenger in sensory transduction
inside the sugar and salt receptor cells.
COMPARATIVE STUDY ON SUGAR TASTE RECEPTOR
PROTEIN CANDIDATES IN_THE FLY.
M. Ozakil, ~. Amakawa*, K. Ozaki! and F.
Tokunaga’, ~Dep. of Bigl BacemOte seis,
Osaka Univ., Osaka and“Dept. of Sci. for
Human Enwironment., Fac. of Human Develop-
ment and Sci., Kobe Univ., Kobe, Japan.
Taste organ in the labellum of the fly
is in the shape of chemosensillum, which
shows less complicated structure than the
taste bud in vertebrate. The chemosensil-
lum includes four functionally differenti-
ated taste cells. One of them, called the
sugar receptor cell, was electrophysio-
logically proposed to have two different
types of sugar receptor site, the furanose
(F site) and the pyranose sites (P site).
We previously found that starch and
levan selectively compete with the stimula-
tive sugars for the P and the F sites, re-
spectively. By using these polysaccharides
as the affinity ligands, we could isolate
the candidate proteins for these sites from
the labellar extract on the native gel.
These proteins showed the consistent
sugar-binding specificities and affinities
with the corresponding receptor sites, re-
spectively. They were also detected in the
extract of the isolated chemosensillum,
which exclusively includes the sensory
processes of the taste cells as cellular
components. The F site candidate protein
was estimated to be 27kDa and the P site
candidate protein which forms a single spot
on the native gel separated into the twin
spots of 31 and 32kDa with SDS-PAGE.
RESPONSIVENESS OF XENOPUS CHEMORECEPTORS
TO BITTER SUBSTANCES.
S. Yamashita. Dept. of Biol., Coll. of Lib.
Arts, Kagoshima Univ., Kagoshima.
Integrated gustatory responses of the
glossopharyngeal (IX) nerve to various
alkaloids and dipeptides were recorded in
the aquatic toad, Xenopus laevis.
Responses to three alkaloids except for
brucine, which showed the estimated
threshold at 10 M, gwere first produced
approximately ax 10 M and tended to
saturate at 10 M. Cross adaptation
coefficients calculated from possible six
pairs of four alkaloids at concentrations
chosen to produce equal neural responses
suggested that both receptor domains which
are stimulated by strychnine-HCl and
quinine-HCl are similar or mostly duplicate,
being rather different from those for
papaverine-HCl and brucine. When pH values
of four alkaloids of a given concentration
were changed by adding HCl, each response
magnitude for three alkaloids except for
papaverine-HCl decreased with decreasing
pH, reducing to 50 % level of the maximal
response magnitude at pH 3. Of fifty
dipeptides used five of strong stimulatory
effectiveness were Pro-Trp, Gly-Arg, Pro-
Tyr, Lys-Tyr and Arg-Ile, which contained
hydrophobic or basic C-terminal amino acids,
Present results suggest that hydrophobicity
of the stimulating substance may be
responsible for an interaction between the
receptor domain and the bitter stimulus.
THE EMETIC RESPONSE OF TELEOSTEAN FISH.
T. Naitoh and T. Kitayama. Dept. of Biol.,
Shimane Univ., Matsue.
From the standpoint of the comparative
study of emesis, we examined the vomiting
ability of fish.
Both Silurus asotus, which has a large
stomach, and Plotosus lineatus, which
lacks a stomach, vomited in response to the
intraperitoneal injection of centrally
acting apomorphine-HCl and copper sulfate
in association with bending of the torso
and pumping of the opercula. Ina
subsidiary study, Odontobutis obscura
also responded to apomorphine-HCl.
Direct administration of copper sulfate
into the stomach in S. asotus and into the
upper part of the intestine in P. lineatus
caused vigorous vomiting. Metoclopramide
monohydrochloride effectively inhibited
apomorphine-induced vomiting and vomiting
by stomach stimulation with copper sulfate
in S. asotus. However, the drug was not
effective in inhibiting either apomorphine-
induced or stomach-stimulated vomiting in
P. lineatus.
The effectiveness of apomorphine-HCl and
the bending behavior that may raise intra-
abdominal pressure suggest that fish are
armed with the same mechanism of vomiting
as in the higher vertebrates. The stomach
is not the necessary factor to develop the
emetic ability in fish. There may be a
species difference in the effectiveness of
antiemetics.
Physiology 1247
ENCAPSULATION OF BRAINLESS PUPAE OF THE
ERI-SILK WORM, SAMIA CYNTHIA RICINI
S. Takahashi. Dept. of Biol., Nara Women's
Univ., Nara.
60-day-old brainless pupae of S.cynthia
ricini contained only about 800 hemocytes
per pl. Plasmatocytes (88%) and granular
cells(12%) were identified. Encapsulation
in the brainless pupae were examined. When
Silicon oi1(10p1) as a foreign object was
injected into the hemocoel, it was encap-
sulated with a thin, smooth membrane, which
was presumably derived from hemolymph com-
ponents. The capsule comprised an inner
amorphous layer and an outer layer of
flattened cells. SEM studies showed that
the primary reactions were the deposition
of coating substance on the foreign sur-
face. During the inner layer formation,
involvement of hemocytes were observed.
When the foreign surface contacted with
the wall (basement membrane) of organs,
such as gut or fat bodies, the deposition
of coating substance occurred on the
surface of the organs. Sessile hemocytes
were also covered with the substance. This
coating layer developed to the inner layer
of capsule. In developing adults, typical
cellular enapsulation was detected. Thus,
it was revealed that cellular responses to
the foreign object were preceded by
humoral responses in the brainless pupae.
MODIFICATION OF GROWTH AND DIFFERENTIATION
OF MOUSE BONE MARROW-DERIVED MAST CELLS
CULTURED WITH GANGLIOSIDE GMs;
H.Fujimaki’, N.Katayama? and K.Nohara’.
‘Environ. Health Sci. Div., Natl. Inst. for
Environ. Studies, Tsukuba and *Dept. of Otorh
inolaryngol. Jikei Univ. Sch. of Med., Tokyo.
The characteristics of mouse bone marrow-
derived mast cells (BMMC) were analogous to
the mucosal type of mast cells. To examine
the effects of ganglioside GMs on growth and
differentiation of BMMC, mouse bone marrow
cells(BMC) were cultured with various concen-
trations of GMs for 3 wk in the presence of
WEHI-3 conditioned medium. Although the nun-
ber of total non-adherent cells was not af-
fected, the number of alcian blue positive
cells(mast cells) was significantly decreased
at 10 and 20 wM GM3. The addition of GM;
for various time intervals revealed that the
decreased number of total non-adherent cells
and the increase of histamine content were
observed in the group added 2 weeks after
initiation of culture. In the growth of BMMC
induced by IL-4, a dose-related suppression
was observed. However, histamine content in
BMMC cultured with IL-4 showed no difference
between GMs-cultured and control groups.
These results indicated that the addition
of GMs to the culture of BMC altered the
growth and differentiation of BMMC.
NEONATAL TOLERANCE IN THE DEVELOPING THYMUS:
CLONAL ANERGY AND CLONAL DELETION
M.Hosono, S.Ideyama and Y.Katsura. Dept.of
Immunol, Chest Dis. Res. Inst, Kyoto Univ. Kyoto.
VB6+ T cell receptor-bearing autoreactive T
cells once-developed in the newborn thymus
of Mlsa antigen-bearing mice started to de-
cline at day 3 of life,and disappeared with-
in the next few days.To understand cellular
basis of self-tolerance, we employed the neo-
natal tolerance system by injecting hemato-
lymphoid cells from Mlsa mice into Mlsb new-
born mice.Mechanisms of unresponsiveness of
Vf6+ cells in the one-week old thymus were
different among anatomical sources of the
injected cells, though cells of bone marrow
(BM), spleen, thymus and perioneal cavity(PC)
cells induced unresponsiveness to the Mlsa
deteminants. At this stage of tolerance, BM
cell-induced toleance was by clonal anergy,
while PCcell-induced toleance was by clonal
deletion. This suggests that BM cells lack
a type of cells which cause clonal deletion
in the one-week-old thymus, though they con-
tain a precursor of deletion-inducing cells
which develope by the second month of age:
Immunohistological data shows that deletion
of VB6+ cells is associated with the intra-
thymic presence of donor-derived B cells.
ANALYSIS OF TRANSPLANTATION TOLERANCE INDUCED IN
METAMORPHOSING XENOPUS TADPOLES — IV
S.Tochinai, M.Ono, S.Tozaki and F.Kobari
Zool. Inst., Fac. Sci., Hokkaido University, Sapporo
The J-strain (JJ) clawed frog, Xenopus laevis, can
easily be made tolerant against semixenogeneic (X. laevis
x X. borealis; JB) adult skin grafted onto larvae before
stage 57 in the presence of the thymus. Tolerance was
never induced to X. borealis (BB) skin. However, when
the BB skin was grafted simultaneously with JB skin,
rejection of BB skin was retarded and tolerance inducibili-
ty to JB skin was affected in turn. The results were corre—
spondent to the size of the BB skin grafted with 2x2 mm?
JB skin: when 2x4 mm? BB skin was grafted all of the BB
and JB skin were rejected acutely, while no JB skin was
rejected when 1x1 mm? BB skin was used (most of BB
skin rejected subacutely), intermediate results were ob-
tained with 2x2 mm? BB grafts. It seems that the immune
reaction against BB skin perturbed tolerance induction to
JB skin, indicating that the tolerance was induced in
larvae after an abortive attack to B haplotype antigens.
The spleen or the thymus removed from the toler—
ant frog was grafted to the immunologically incompetent
thymectomized frogs carrying previously grafted JB skin.
Skin graft rejection initiated in the thymus-—grafted frogs,
while the spleen-grafted frogs showed uninterrupted
acceptance of JB skin. Although the presence of the
suppressor cells in the tolerant animals is not perfectly
demonstrated, it is highly probable that the suppressor
activity resides in the tolerated JB skin and the spleen of
tolerant animals. Attempts are being made to establish
the cellular basis of the tolerant state.
1248 Physiology
ROLE OF THE PROLIFERATING SPLENOCYTES IN IMMUNE
RESPONSE OF NORMAL AND TOLERANT XENOPUS LAEVIS
J. Sakuraoka and S. Tochinai. Zoological Institute,
Faculty of Science, Hokkaido University, Sapporo
We have established a simple and reliable method
to quantify the proliferating cells in vivo and jin vitro by
using 5'-Bromo-2'-deoxyuridine (BrdU) and anti-BrdU
antibody in Xenopus laevis. When the adult or larval J-
strain (JJ) frogs were grafted or injected with
semixenogenic (X. laevis x X. borealis, JB) cells, splenic
lymphocytes proliferated even in the frogs tolerant to JB
skin. In order to examine the role of proliferating cells in
these frogs, splenic lymphocytes from normal and tolerant
animals stimulated with JB cells were transferred to the
immunoincompetent thymectomized frogs previously
grafted with JB skin. In frogs injected with splenocytes
from normal frogs, the JB skin graft rejection initiated
promptly and ended in about 10 days, while it took longer
in the frogs injected with splenocytes depleted
proliferating cells by BrdU incorporation and fluorescent
light treatment, indicating that the proliferating cells were
actively involved in the rejection process.
On the other hand, the results were not so simple
in thymectomized frogs injected with tolerant splenocytes.
Interestingly, the tolerant status was broken down in many
of the frogs when received splenocytes from JB cell
stimulated tolerant frogs, while the tolerance was not
broken down in many of the frogs injected with BrdU-light
treated tolerant splenocytes. It is suggested that even in
the tolerant frogs effecter cells were proliferating in
response to the toleragenic JB cells along with the
possibly participating suppressor cells.
OBSERVATIONS OF HEMATOPOIETIC SYSTEMS IN
RANA CATESBEIANA, BY SEM.
N.Mimori and K.Sugiyama. Dept. of Biology,
Fac. of Sci., Hirosaki Univ., Hirosaki 036
We examined the hematopoietic systems
during metamorphosis of anuran amphibia Rana
catesvbeiana, usng transparent and/or
scanning electron microscopy. Following
findings are demonstrated that ():the surface
-profiles of hematopoietic cells were
fundamentally same as that shown in
mammalian cells. @:the phagocytosis against
non-self antigen was obseved by neutrophils
and by macrophages. @:characteristic inner
surface features were demonstrated by the
ion ecthing treatment-method of SEM. char-
acteristically in neutrophils and eosino-
phils. @:the mesonephros may function as a
supply-source for leukocytes of larvae,
however, the spleen may perhaps function as
the bone marrow during the all-life of this
anima) similar to marmmmalia.@i:thehypertrophy
of bone marrow was clearly obseved at the
middle stages of metamorphosis, and
continued till the end of metamorphosis.
THE LINING CELLS OF HEMOCOEL PARTICIPATE
IN INTERNAL, DEFENSE OF,LAND SLUG
K. Yamaguchi , E.Furuta agd A.Shimozawa
Lab. of Med. Sci. and “Dept. of Anat.,
Dokkyo Univ. Sch. of Med., Tochigi.
When microorganisms invade into the
hemocoel of land slug, Incilaria
fruhstorferi, the number of hemolymph
cells increase in the hemolymph and then
they encapsulate and phagocytose the
invaders. The lining cells of the
hemocoel, blood vessel and heart walls
of the land slug were observed by SEM
and TEM. Only the lining cells of hemocoel
wall possessed numerous microvilli, high
electron dense nuclei and basal lamina.
These cells protruded and freed from
the wall into the lumen, when non-self
materials(yeasts) were inoculated into
the hemocoel. They were morphologically
similar to macrophages. Otherwise in
tissue culture, the migrated cells from
explants of hemocoel wall mainly consist
of fibroblast-like cells and macrophage
like cells and these cells phagocytosed
yeasts. By histochemistry, the circulating
hemolymph cells were strongly positive
to non-specific esterase reaction, while
two kinds of the migrated cells were
weakly positive. These results suggest
that the hemolymph cells may be derived
from the hemocoel wall and the
fibroblast-like cells may be able to
transform to macrophage-like cells.
LECTINS FROM BODY SURFACE MUCUS OF THE
LAND SLU 2 1
-Furuta’ , T.Takagi” and A.Shimozawa
Dept. of Anat., Dokkyo Univ. Sch. of
Med. Tochigi, and Biol. Inst., Fac.
of Sci., Tohoku Univ., Miyagi
The characteristics of lectin activity
have been shown to be exhibited by mucus
substances from a variety of biological
sources. In Incilaria fruhstorferi, well
developed mucus glands and goblet cells
are often found under or in the epidermis.
The mucus agglutinated human A and B
erythrocytes and the hemagglutination
was specifically inhibited by low
concentration of N-acethyl-galactosamine.
All of this activity was completely
inactivated by heating at 56°C for 30
min. By a CM-cellulose chromatography
and a reverse phase HPLC, three small
lectins (Y1l5a,b,c) from the mucus were
purified. The molecular weight of Yl15a,b
and c was 15,16 and 17kDa, respectively,
by SDS-PAGE analysis. The amino acid
sequence of these proteins was determined
by peptide sequences analysis. Although
complete sequences were not established
yet, the sequences of these proteins
were very similar (60-70% identical).
Especially positions of 6 cysteine
residues were very conservative in three
proteins and these cysteines constructed
three disulfide bonds in each molecule
which were typical in C-type lectin
domain. These results suggest that Yl5a,b
and c belong to C-type lectin.
Physiology 1249
SPECIES DIFFERENCES OF PHAGOCYTIC
ACTIVITY IN FISH ENDOCARDIAL CELLS.
H.Nakamura’ , S.Kikuchi* and A.Shimozawa.
‘Dept. of Anat., 7Dokkyo Univ. Sch. of
Med., Tochigi, Kominato Lab., Fac. of
Sci., Chiba Univ., Chiba.
Endocardial (EC) cells of some species
of fishes were reported to be phagocytic
however all the reports on this
phenomenon have been given by in vivo
studies. We observed in vitro phagocytic
activity of EC cells of several species
of fishes. Hearts of fish were dessected
and incubated organotypically in the
tissue culture medium DM-170 containing
1% Perikan ink (carbon particles) for 2
hours at about 23°C. They were observed
histologically. Animals used in this
study are as follows; medaka (Oryzias
latipes), guppy (Poecilia reticulatus),
neon tetra (Paracheirodon innesi), lemmon
tetra (Hyphessobrycon pulchripinnis),
goldfish (Carassius auratus) and rose
bitterling (Rhodeus ocellatus). EC cells
of cyprinodontiformes (medaka and guppy)
showed intense phagocytic activity. EC
cells of cypriniformes (goldfish and
bitterling) showed moderate and those of
characiformes (neon tetra and lemmon
tetra) showed weak or almost no
phagocytic activity against carbon
particles. Phagocytic activity of EC
cells may relate to the phylogenetic
status in self defense system of fish.
INTERACTION BETWEEN CELL TYPES IN
HEMOCYTE CLOTTING SYSTEM OF THE SPINY
'LOBSTER, PANULIRUS JAPONICUS.
H. Aono. National Research Institute of
Aquaculture, Mie.
In order to clarify cell type specific
roles in the hemocyte clotting system,
hemocytes isolated from hemolymph of the
spiny lobster (Panulirus japonicus) were
separated into two populations, i.e.,
semigranular and hyaline cells (SGH
eells) and granular cells by density
gradient centrifugation. Clotting ability
of each hemocyte population was tested in
vitro. Whole hemocytes formed a cellular
elot when incubated with some kinds of
bacteria or lipopolysaccharides (LPS),
while SGH cells did not clot nor show
morphological change during incubation
with LPS. Granular cells were observed to
degranulate when exposed to LPS but
clotting did not occur. However, medium
conditioned with both granular cells and
LPS strongly induced clotting of SGH
cells. Lysate of granular cells also
induced clotting of SGH cells.
Microscopic observation showed that the
lysate acts on SGH cells as a lytic
factor. These results suggest that
granular cells release a factor in
response to LPS, and the factor induces
lysis and clotting of SGH cells.
THE NATURE OF MARKERS FOR RECOGNITION ON
HEMOCYTES IN ALLOGENEIC PHAGOCYTOSIS OF THE
ASCIDIAN, HALOCYNTHIA RORETZI
S.Ohtake, T.Abe, F.Shishikura, and K.Tanaka
Department of Biology, Nihon University
School of Medicine, Tokyo.
Small granular amebocyte (SG) phagocy-
toses each other in allogeneic mixed cul-
ture of the hemolymph of H. roretzi in
vitro. We named this response "allogeneic
phagocytosis". When washed hemocytes were
cultured with glutaraldehyde-fixed hemo-
cytes for 30 min, only 2-5% of SGs phago-
cytosed the fixed autogeneic hemocytes
(fAutoHc) but 60-80% of SGs did the fixed
allogeneic hemocytes (fAlloHc). To examine
the recognition mechanism, we treated the
target cells, fAutoHc and fAlloHc, with
B-galactosidase or trypsin before mixing
with washed SGs, or added GalNAc in the
mixture of SGs and targets. The treatment
of fAutoHe with B-galactosidase (10-100
pg/ml) increased the number of SGs phagocy-
tosing them from 4% to 48.2-68.7%. The
addition of GalNAc (0.3-30 pg/ml) decreased
the number of SGs phagocytosing fAlloHc
from 70% to 19.6-11.2% without any sup-
pression of the phagocytic ability of SGs
against latex beads. The treatment of
trypsin (0.01-1mg/ml) decreased the phago-
cytosis against fAlloHc although it did
not affect fAutoHc. These results sug-
gest that SGs may see specific glycoprotein
markers on the surface of hemocytes to
distinguish allo-cells from auto-cells and
phagocytose allo-targets.
UNIQUE AGGREGATE FORMATION BY A HEMOCYTE OF
TUNICATE, STYELA CLAVA.
1 and
T.Sawada , S. Tomonaga®, T. Fukumoto
.L. Cooper3°. ‘Dept. Anat., Sch. Med.,
Sch. Allied Health Sci,, Yamaguchi Univ.,
Ube, Yamaguchi, and 3hept. Anat. Cell
Biol., Med. Sch.,Univ. California, Los
Angeles (UCLA), California, USA.
In tunicates, endothelial cells which
seal blood vessels are absent and hemocytes
may include connective tissue cells with
functions other than those of vertebrate
hemocytes. After culturing hemocytes of
Styela clava, we found unique aggregate
formation by hemocytes which consisted of a
homogeneous hemocyte type (basophilic
granulocytes). These granulocytes became
associated and spread on matrices in oblong
shape, adhering to each other and forming a
flat sheets initially. These sheets became
folded into a round mass which was
consisted of homogeneous cell types,
tightly compacted as if they were in the
process of forming tissue. The nature of
these granulocytes suggested that they are
related to mesenchymal cells with
epithelial or fibroblastic characteristics,
and not those associated exclusively with
immunodefence as for other hemocytes. A
monoclonal antibody UB15, which reacts to a
sub-population of hemocytes of Ciona
savignyi and Halocynthia roretzi, exhibited
a specific staining against this basophilic
granulocytes.
1250 Physiology
DESALINATION IN THE ESOPHAGUS OF THE SEA-
WATER EEL
K.Nagashima and M.Ando. Lab. of Physiol.,
Fac. of Integrated Arts & Sci., Hiroshima
Univ., Hiroshima.
The esophagus of the seawater teleost
desalts the swallowed seawater. However,
the mechanisms of the desalination is not
clear yet. To clarify the mechanisms, net
Na’, Cl. and water fluxes were measured
simultaneously under various conditions.
Following’osmotic gradient, water moved
without accompanying significant Na’ and
Cl ions; the calculated osmgtic
permeability being 3-4 x 10 ml/cm¢.s.0Osm.
This value is lower than that in "tight
epithelia" such as frog skin and urinary
bladdeg, though the tissue resistance of 70
ohm.cm“ belongs to "leaky epithelia". By
altering Na* and Cl concentrations in the
mucosal fluid, while the serosa was bathed
with normal Ringer solution, a relationship
between ion flux and its driving force was
obtained. Both Na andCl fluxes were
related almost linearly to their electro-
chemical gradients across the esophagus,
suggesting passive movements of Na and Cl
However, most of these ion fluxes were
coupled each other. Moreover, the couple
NaCl transport was jnhibited by NaCN (10.
M) and ouabain (10 * M), suggesting in-
volvement of some active processes in this
coupled transport. The coupled NaCl trans-
port may be due_to NaCl cotransport, since
bumefanide (10 ° M) and hydrochlorothiazide
(10°* M) inhibited the NaCl transport.
Effects of hypoxia on renal function in carp
I. Kakuta and S. Murachi. Dept. of Biotechnology,
Senshu Univ. of Ishinomaki, Ishinomaki.
The influence of hypoxia upon renal function in
fish has been studied, and investigation of blood
properties and cardiac function was conducted to gain
a better understanding of renal response to hypoxia
in fresh water teleosts.
Hypoxic conditions were induced by allowing the
fish to consume available dissolved oxygen (D0) in a
10 1 experimantal tank. When the DO level fell to
about 5% of oxygen saturation after several hour
stress, carp had lost balance. As water flow and
aeration to the tank were established, carp recovered
their balance within several minutes.
Glomerular filtration rate (GFR) and urine flow
decreased with increased urinary concentrations of
bio-components, except protein, in the course of
hypoxia. Decreases in blood pH and plasma prosta-
glandin Ee» ,and increases in Ht, plasma K*, Ca**,
Mg**, Pi, ammonia, lactic acid and catecholamines
were observed as hypoxia progressed. Decreases in
heart rate (HR) and blood pressure in dorsal aorta
(BPd), and increase in the duration of electrical
systole in electrocardiogram were also observed.
Increased GFR and urine flow, and higher values
for urinary components, except protein, compared with
those of the control were found in the initial post-
stress stage. Increases in plasma angiotensin II,
HR and BPd were also observed. They resumed to the
resting levels after 12hr.
IMMUNOCYTOCHEMICAL LOCALIZATION OF GLUT1,
GLUT4, AND THEIR CHIMERIC GLUCOSE
TRANSPORTER PROTEINS.
K.TAKATA*, T.ASANO?, Y.OKA?, AND H.HIRANO?*.
*Dept of Anatomy, Kyorin Univ. Sch. of Med.
Mitaka,Tokyo, *3rd Dept. of Int. Med., Fac.
of Med., Univ. of Tokyo, Tokyo.
Facilitative glucose transporter
isoforms, GLUT1 and GLUT4, have different
intracellular distributions despite their
very similar structure. GLUT1 is found at
the plasma membrane of various tissues and
cells, while GLUT4 is localized in the
cytoplasmic compartments, such as Golgi
apparatus in adipocytes and muscle cells.
To elucidate the differential targeting
mechanisms, GLUT1, GLUT4, and GLUT1/GLUT4
chimeric glucose transporters were stably
expressed in Chinese hamster ovary cells.
These cells were subjected to
immunofluorescence and immunoelectron
microscopy as well as measurement of
glucose transport activity. GLUT4 was
mostly localized intracellularly,
especially in the trans-Golgi regions,
while GLUT4 was found at the plasma
membrane. These results suggest the
existence within GLUT molecule of domain(s)
responsible for proper intracellular
targeting. The results of chimeric
glucose transporters revealed that two
domains of GLUT4, which are not N-terminal
or C-terminal domain, determine its
targeting to the intracellular
compartments.
IMMUNOCYTOLOGICAL DETECTION OF VACUOLAR
TYPE H'-ATPASE FROM THE VANADOCYTES IN
ASCIDIANS
T. Uyama and H. Michibata, Mukaishima
Marine Biol. Lab., Hiroshima Univ.,
Hiroshima.
Level of vanadium in vanadocytes of
Ascidia sydneiensis samea is in excess
of 10 mM, which corresponds to a level
more than million times higher than
that in sea water. However, it is
unclear that the energetistic mechanism
of the accumulation of vanadium against
this large gradient of concentration.
Since the vacuolar contents of vana-
docytes show very low pH values of
approximately 2, we have examined
whether vacuolar type H —ATPase exists
in the vacuolar membranes, to consider
a possibility of the energestistic
conjugation of the ATPase with the
accumulation of vanadium.
As the result, immunocytological
analysis revealed that the antisera
specific to 72 kDa and 57 kDa subunits
of vacuolar type H’-ATPase reacted with
vanadocytes of A. sydneiensis samea.
Immunoblotting analysis showed that the
antigens recognized by the antisera
were 70 kDa and 57 kDa proteins, re-
spectively.
RAISING OF MONOCLONAL ANTIBODY AGAINST
BLOOD CELLS OF VANADIUM-RICH ASCIDIAN,
ASCIDIA AHODORI
J. Wuchiyama, T. Uyama and H. Michiba-
ta, Mukaishima Marine Biol. Lab.,
Hiroshima Univ., Hiroshima.
Blood cells of vanadium-rich ascid-
ians can be classified into eleven
types but the relationships between
different types of blood cells has not
been proven in spite of many cytologi-
eal and morphological studies.
We, therefore, intend to raise mono—
clonal antibodies against several types
of blood cell to follow the lineage of
these cells.
Among several types of blood cell in
Ascidia ahodori, signet ring cells was
separated using percoll density gradi-
ent centrifugation and the subpopula-
tion was injected intraperitoneally to
balb/e mouse to immunize.
The raised monoclonal antibody
(AKBB3) specific to the signet ring
cells of A. ahodori recognized the
Signet ring cells of not only A. ahodo-
ri but also A. sydneiensis samea.
Molecular weight of antigen reacted
with the antibody was estimated at
about 77 kDa in A. sydneiensis samea
and that in A. ahodori is under inves-—
tigation.
SPONGE SPICULES IN THE BODY OF SPONGIVOROUS
SEA URCHINS
R.Birenheide!, T.Motokawa! and S.Amemiya2. IBiol. lab.,
Tokyo Inst. of Technology, Tokyo, 2Misaki Mar. Biol.
Station, Miura-shi, Kanagawa.
Some sea urchin species feed on sponges but nothing is
known about the possible problems caused by penetrating
sponge spicules. We screened the feeding tract, coelomic
organs and skeleton of the species Asthenosoma ijimai,
Araeosoma owstoni, Hapalosoma gemmiferum, (Echinothu-
rioida) and Diadema setosum (Diadematoida). By dissolving
tissues or skeleton and subsequent filtration silicate sponge
spicules were isolated from these sea urchins. Spicules of two
types, i.e. raphides and styles were found. Histological
sections showed the distnibution of spicules in the tissues. In
the feeding tract especially pharynx and esophagus contained
more raphides than styles. No specific tissue reactions on the
sponge spicules were observed. In the coelom, raphides were
accumulated in Stewart Organs, gills and in or near the
ampulla. They were always entangled by brown bodies.
Brown bodies are the main carrier of wastes in sea urchins.
They leave coeloms by penetration through epithelia. The
accumulation of brown bodies containing sponge spicules
suggests that the spicules are too Jong to be transported
through epithelia and thus are stored in coelomic cavities.
From plates and spines we could isolate more styles than
raphides. The spicules were often incorporated into the
skeleton of the sea urchins.
Physiology 1251
TWO NOVEL-FAMILY PEPTIDES ISOLATED FROM THE
ABRMs OF Mytilus.
Y. Fujisawal, 1. Ikedal, yY. Muneokal, o.
Matsushima2 and I. Kubota3. Fac. of
Integrated Arts and Sci., Hiroshima Univ.,
Hiroshima, 2Inst. of Biol., Fac. of Sci.,
Hiroshima Univ., Higashi-hiroshima and
Suntory Bio-Pharma Tech Center, Gunma.
Two contractile peptides were isolated
from the anterior byssus retractor muscles
(ABRMs) of the bivalve mollusc Mytilus
edults. Their structures were determined
to be as follows:
Peptide 1 GPFGtTH HMI K amide (GPFG-8)
Peptide 2 GPFGLWN KH G amide (GPFG-9)
Both peptide 1 (octapeptide) and 2
(nonapeptide) were found to have Gly-Pro-
Phe-Gly- at their N-terminal parts as a
common structure, and hence they were
designated GPFG-8 and GPFG-9, respectively.
The peptides do not appear to be members of
any other previously identified peptide
family. Both the peptides evoke a weak
contraction in the ABRM. However, the ABRM
shows tachyphylaxis to the peptides; the
second contractile response of an ABRM to
the peptides is far smaller than the first
response. Therefore, the peptides seem not
to be physiological excitatory neuro-
peptides in the muscle. They might play a
role for keeping a homeostatic state of the
muscle.
In addition to the above peptides, three
other peptides were newly isolated in this
study. They were two Mytilus inhibitory
peptide analogues and a related peptide.
RELATIONSHIP BETWEEN ERYTHROPOIETIN AND
ERYTHROID COLONY-STIMULATING ACTIVITY IN
MOUSE PLASMA.
$.Sakata!, Y.Enoki! and M.Ueda’.*Dept. of
Physiol., Nara Med. Univ., Kashihara, Nara
and *Research Inst. of Life Sci., Snow
Brand Milk Products, Tochigi.
A relationship between erythropoietin
(EPO) and erythroid colony-stimulating
activity (ECSA) in mouse plasma was exa-
mined in fetal mouse liver cell (FMLC)
cultures using a monoclonal antibody (MoAb)
R2 raised against recombinant human EPO.
Most of the ECSA in plasma from normal,
anemic and hypoxic mice was neutralized by
MoAb. This neutralization could be reversed
by addition of excess of anemic plasma or
by preincubation of MoAb with goat anti-
mouse IgG antibody. Most of the plasma ECSA
was bound to an immunoadsorbent column
containing the immobilized MoAb, and the
retained ECSA was completely neutralized by
MoAb. The plasma ECSA and standard EPO
showed parallel dose-response curves and
additive effect on CFU-E stimulation. Based
on these findings, we conclude that mouse
plasma ECSA detected by the present CFU-E
assay using FMLCs is mainly due to EPO.
1252
OXYGEN TRANSPORT BY HEMOGLOBIN IN DAPHNIA
MAGNA.
Michiyori Kobayashi!, Kenichi Ishigaki?,
Michisuke Kobayashi? and Kiyohiro Imai’.
1Department of Biology, ?Information
Processing Center, 3Department of
Physics, Faculty of Science, Niigata
University, Niigata, and 4Department of
Physiology, Medical School, Osaka Univer-
sity, Suita, Osaka.
Using Hb from Hb-rich (Hbr) and Hb-
poor (Hbp) Daphnia magna, curve-fitting
Was carried out for the oxygen equi-
librium curves, and Adair constants were
determined.
The oxygen saturation (Y) of hemo-
globin Hbr and Hbp in Adair’s equation
was differentiated by p to obtain the
differential coefficient (Y’). The Y’ apr
/Yuop ratio showed a peak (2.9) at posi-
tion close to pso of Hb from Hb-rich
animals. Using the monocomponent human
Hbs having oxygen-affinity almost equiv-
alent to those from Hb-rich and Hb-poor
animals, a markedly high ratio of dif-
ferential coefficient was obtained. This
indicates that substitutive performance
of the function of high oxygen-affinity
Hb by low oxygen-affinity Hb is very dif-
ficult. It is considered that for adapta-
tion to a wide range of environmental
oxygen condition by means of Hb, abnormal
increase in Hb concentration is avoided
by possession of multicomponent Hb.
ELECTROPHYSIOLOGICAL STUDIES ON BINDING
ACTIVITY OF AZIDE DERIVATIVE OF
TETRODOTOXIN TO Na CHANNELES.
Seiji UEHARA. Dept. of Biology, Saitama
Med. School,Saitama.
Binding activity of an azide derivative
of tetrodotoxin(TTX) to Na channeles of
a sciatic nerve of a bullfrog was electro-
physiologically studied. The derivative
was synthesized by reacting TTX and 1-
fluoro-2-nitro-4-azidophenyl- g alanine,
and purified with HPLC. Biological activ-
ities of the derivative was 1/40 of that
of TTX.
1) The nerve was incubated with the de-
rivative in the dark and then photoirra-
diated with a deuterium lamp. Afetr the
nerve was repeatedly washed with Ringer's
sol., a small decrease in the amplitude
of the action potential was observed. But,
photoirradiation alone also sometimes in-
duced a decrease. Therefore, it could not
be concluded that the small decrease was
caused by the derivative.
2) Instead of photoirradiating directry
the nerve, the derivative was preliminar-
ily photolyzed and the nerve was incu-
bated with the photolyzed derivative
changing incubation time. Incubation for
60 - 90 min. induced strong inhibition of
recovery of the amplitude after washing
repeatedly the nerve. From these results,
it can be concluded that the derivative
binds covalently to Na channeles.
Physiology
GTP-BINDING PROTEIN IN RHABDOMERIC
PHOTORECEPTORS.
T.Suzuki!, K.Nagai’, K.Narita2, Y.Kito2, M.Michinomae$
and K.Yoshihara*. ‘Dept. of Pharmacol., Hyogo
Coll. Med., Nishinomiya, @Dept. of Biol., Fac. of Sci.,
Osaka Univ., Osaka, 3Dept. of Biol., Konan Univ., Kobe
and 4Suntory Inst. of Bioorg. Chem., Osaka
We prepared polyclonal antibodies against
polypeptides of the sequences of cholera toxin and
pertussis toxin binding sites of bovine transducin and
N-terminal region of Drosophila Gaq (Pab CTX, Pab
PTX and Pab DGqN). Pab DGQN strongly reacted with
42kD protein localized to the head of Drosophila. We
carried out immunoblot analysis to identify the G-protein
of rhabdomeric photoreceptors. The Pab DGqN-positive
42kD protein was detected in rhabdomal fractions of
crayfish, shrimp, octopus and squid but not in frog and
bovine rod outer segments. The 42kD protein was
solubilized with detergents and purified with Con A-
and DEAE-columns. The purified protein was associated
with 35kD protein which is positive to the antibody
against GB. Pab CTX and Pab PTX did not react with
the squid 42kD protein. These results indicate that
major G-protein in rhabdom of photoreceptor is Gq-type.
The Pab DGqN-positive 42kD protein was found also in
water-soluble fraction of squid retinal homogenate. This
suggests that Gaq exists in water-soluble and
membrane-bound forms.
PHOTOSENSITIVE STAGES IN PHOTOPERIODISM
FOR WING FORM AND REPRODUCTION IN A WATER
STRIDER, AQUARIUS PALUDUM.
T. Harada. Dept. of Biol., Fac. of Sci.,
Osaka City Univ., Osaka.
Long-day photoperiods induced short-
winged form and reproduction, short-day
caused long-winged form and diapause, and
the step-wise decrease in photophase
(by 15 min every 5 days) from 14.5 h to
13.5 h before the last (fifth) instar
stage induced diapause in Aquarius paludum
(Harada and Taneda, 1990). The step-wise
decrease in the same way from 15 h to 14 h
caused the long-winged form, but did not
induce diapause. Insects were reared
under a short-day of 9.5L-14.5D except
each specific stage (12-13 days) under a
long-day photoperiod of 15.5L-8.5D, i.e.,
during the first and second instars (group
A), the third and fourth instars (group B),
or the fifth instar (group C). The pro-
portion of long-winged form was more than
85% in all the groups. 18.2% of the
females laid eggs in group C. All the
females in group A and B entered diapause.
However, there was a difference in the
form of the ovarioles between each of
groups A, B and the group of females
reared under the short-day through the
nymphal stage. The sensitive stages for
wing form and reproduction seem to be in
the wide range of the first to fourth
instar and the whole nymphal stage,
respectively.
Physiology
CIRCADIAN OCULAR RHYTHMS IN THE JAPANESE
QUAIL (COTURNIX COTURNIX JAPONICA) AND THE
EFFECT OF MELATONIN INJECTION.
M.Sasaki and T.Oishi
Dept. of Biol., Nara Women's Univ., Nara
We investigated circadian rhythms of
retinal melatonin, mitotic rate in the
corneal epithelium and visual pigment
production in the retina, and the effects
of melatonin injection on the rhythms were
also observed.
Retinal melatonin was high in darkness
and low in light under LD cycle (LD 12:12),
and the rhythm continued in continuous
darkness (DD), but seems to disappear in
continuous light (LL). The rhythm of
mitotic rate in the corneal epithelium was
very Similar to the rhythm of melatonin.
The outer segments of rod cells were
stained intensely by the rhodopsin anti-
serum throughout 24 hours in LD cycle, LL
and DD. However, some parts of inner
segments (Golgi area) were stained only
during light phase of LD cycle. The rhythm
seems to disappear in constant conditions
(high immunoreactivity throughout 24 hours
in LL, and intermediate throughout 24 hours
in DD).
When melatonin was injected into the
right eye at 8:00 for 5 days in LD 12:12,
a distinct phase shift was induced in the
corneal mitotic rhythm compare to the
rhythm in the saline injected left eye, but
the eects on the visual pigment
production seem to be meagre.
SPECIES SPECIFICITY OF HIBERNATION
RELATED PROTEINS (HPs) IDENTIFIED IN
PLASMA OF CHIPMUNKS.
N.Kondo, H.Honda and J.Kondo’. Mitsubishi kasei
Inst. of Life Sci., Machida, Tokyo, ‘Mitsubishi
Kasei Corp. Res. Center, Yokohama.
In our previous studies, novel types of protein
have been identified in plasma of a mammalian
hibernator, chipmunks (Tamias asiaticus). These
proteins (HP-20, 25, 27 & 55) were specifically
reduced or disappeared from the blood during
hibernation under constant laboratory conditions. In
the present study, the detection of HP-20s in the
blood of various species of rodents was carried out
using monoclonal antibodies (mouse) and antisera
(rabbit) to HP-20s. In immunoblotting analysis, the
existence of proteins reacting with the monoclonal
antibodies and antisera was shown in plasma from
ground squirrels (Citellus tridecemlineatus) and
marmots (Marmota flaviventris), both of which are
hibernating species. In plasma from other species
(tree squirrels, golden hamsters, djungarian
hamsters, mice and rats), these antibodies and
antisera did not recognize HP-20s. From further
immunoblotting studies, at least HP-20 and 25 were
identified in marmots and ground squirrels. The fact
that HPs were detected in two other species of
hibernators indicate the specificity and physiological
significance of HPs for hibernation.
1253
1254 Endocrinology
ORIGIN AND MIGRATION OF LUTEINIZING HOR-
MONE-RELEASING HORMONE (LHRH) NEURONS IN
THE CHICK EMBRYO: A CARBOCYANINE DYE
ANALYSIS.
S. Murakami and Y. Arai. Dept. of Anat.,
Juntendo Univ. Sch. of Med., Tokyo.
Recently we reported that unilateral
olfactory polacodectomy resulted in the
absence of LHRH neurons in the olfactory-
brain axis of the operated side in the
chick embryo. This suggests that LHRH
neurons originate in the olfactory pla-
code, after which they migrate into the
brain.
In the present study, to obtain a
direct evidence for the migration of the
LHRH neurons from the olfactory epithelium
to the brain, the epithelial cells of the
olfactory placode of the chick embryo (EDs
3.5-4) were labeled with Dil. The labeled
embryos were incubated in ovo for 1 to 4
days. DilI-labeled cells were first de-
tected in the olfactory nerve 1 day after
application of Dil. Two-four days after
application of Dil, labelled cells sequen-
tially appeared in the rostral and medial
forebrain, and in the septo-preoptic area.
The distribution pattern of Dil-labeled
cells closely resembled that of LHRH
neurons. Double staining for LHRH and Dil
showed that a part of Dil-labeled cells
co-expressed LHRH immunoreactivity. These
results provide evidence for the actual
migration of LHRH neurons from the olfac-
tory region to the septo-preoptic area.
LATERAL ASYMMETRY OF LHRH CELLS IN
THE BRAIN OF THE MOUSE WITH TESTICULAR
FEMINIZATION MUTATION (T£m/Y) .
Y. Inase, T. Machida and T. Noumura.
Dept. of Regulation Biol., Fac. of
Sci., Saitama Univ., Urawa, Saitama 338.
The X-linked testicular feminization
mutation (Tf£m/Y) in the mouse is charac-
terized by insensitivity of the target
cells to androgens. In the present
experiment, number of immunoreactive
LHRH cells was compared between the
right and the left side of the brain
in Tfm/Y mice.
Although we have already exhibited
that the number of LHRH cells are consist-
ly greater in the right side of the
brain than in the left side in normal
male mice, no lateral asymmetry was
found in the number of LHRH cells in
Tf£m/Y mice.
Removal of testes of both sides in
Tfm/Y mice caused an increment in number
of LHRH cells in the brain. In normal
male mice, however, number of LHRH
cells significantly decreased following
bilateral orchidectomy.
The results demonstrated a unique
behavior of LHRH cells in the brain
of Tfm/Y mice.
SEX DIFFERENCE IN THE DISTRIBUTION
OF LHRH NEURONS IN THE MOUSE BRAIN.
W. Nishido and T. Machida
Dept. of Regulation Biol., Fac. of
Sci., Saitama Univ., Urawa, Saitama 338.
In contrast to known sexual dimorphism
present in the rat brain, no sex differ-
ences have been found in volumes. of
mouse hypothalamic structures. In order
to clarify possible sex differences
of the ‘brain in the mouse, ontogeny
and distribution of immunoreactive
LHRH neurons were examined in male
and female mice.
In male mice, LHRH neurons increased
in number from 7 days of age to 30
days and thereafter decreased drastically.
In adult female mice, number of LHRH
neurons fluctuated during estrous cycle:
largest at proestrus and smallest at
estrus. Number of these neurons in
adult male mice was similar to that
of proestrous females. Neonatal estrogen
sterilization of female mice caused
as adults an increase in number of
LHRH neurons in the brain. The results
exhibited sexual dimorphism of the
number of immunoreactive LHRH neurons
in the mouse brain.
GnRH IMMUNOREACTIVE SUBSTANCE IN RAT PINEAL
GLAND IS A MEMBRANE ASSOCIATED PROTEIN.
M.K. Park, H. Kogo, S. Kawashima, and K.
Wakabayashi~. Zool. Inst., Fac. 0o SGic,
Univ. of Tokyo, Tokyo 113 and Hormone
Assay Center, Inst. of Endocrinol., Gunma
Univ., Gunma 371, Japan
Immunoreactive and bioactive GnRH in
the pineal gland has been reported in
bovine, rat, and ovine. A monoclonal anti-
body, LRH13 is a well characterized GnRH
specific monoclonal antibody (Park and
Wakabayashi .1986). Using this antibody,
strong immunohistochemical signal in the
rat pineal gland was characterized. SDS-
polyacrylamide gel electrophoresis (SDS-
PAGE) and immunoblot were used to charac-
terize this immunoreactive substance.
Immunoblot showed that apparent molecular
weight of the substance is approximately 52
K Dalton (KD). The immunochemical signal of
52 KD protein was GnRH specific because of
the complete blocking with 0.5 mg of GnRH
in 10 ml of LRH13 solution (1,000 times
diluted). The signal was detected in the
membrane fraction of the pineal homogenate,
and not in the cytosolic supernatant.
Treatment with NaCl (140 or 500 mM), Mg**
(5 mM) or EDTA (10 mM) in 50 mM Tris-Cl, pH
7.4 could not release, but Triton X-100 (2
%) released the 52 KD protein from the
membrane fraction of the pineal gland.
These results suggest that the rat
pineal GnRH-immunoreactive substance has a
GnRH-like domain, and is a kind of membrane
associated protein.
Endocrinology 1255
LORDOSIS INHIBITING INFLUENCE IN THE DORSAL
RAPHE NUCLEUS IN MALE RATS: EFFECTS OF
NEURAL TRANSECTIONS.
HORMONAL AND BEHAVIORAL STUDIES OF
WINTERING FLOCK OF PINTAILS (Anas acuta)
M. Wada!. A. Kishida?: R. Tanabe? and K. Fukui3. 1Dept.
Gen. Educat., Tokyo Med. Dent. Univ., Ichikawa, Chiba,
2College of Agr. & Vet. Medicine., Nihon Univ., Fujisawa,
Kanagawa and 3Res. Center, Wild Bird Society of Japan,
M. Kakeyama and K. Yamanouchi.
Dept. of Basic Human Sciences, Sch. of Human
Sciences, Waseda University., Tokorozawa.
To clarify lordosis inhibiting neural
pathways from the dorsal raphe nucleus (DRN)
in male rats, several types of cuts around
DRN were performed and lordosis behavior was
examined. Male rats were castrated and
subjected to ventral-horizontal (VHC),
dorsal- horizontal (DHC), anterior (AC) or
posterior (PC) cut of DRN. Four weeks
after, all animals were implanted with
Silastic tubes containing estradiol, and
then lordosis behavior was observed. Non-
brain surgery control males showed low score
of lordosis quotient (LQ). LQs in either
DHC or PC males were also low, and was
comparable to those in control males. In
contrast, VHC males showed higher LQ value
than control males. As well as VHC males,
AC males displayed high LQ score. These
results suggest that ventral and anterior
Outputs (and/or inputs) of the dorsal raphe
nucleus are involved in the lordosis
inhibiting system in male rat brain.
A Possible Existence of Non-Strial Pathway
of Amygdala in Control of Male Rat Sexual
Behavior.
Y. Kondo and K. Yamanouchi.
Neuroendocrinol. Lab., School of Human
Sciences, Waseda University, Tokorozawa.
We have reported an importance of the
medial amygdala, as well as the medial
preoptic area, in regulation of male rat
sexual behavior. Anatomical evidence shows
that the medial amygdala innervates the
medial preoptic area mainly through the
stria terminalis (ST). In this experiment,
neural transection of the ST (STC) was made
in male rats which were previously castrat-
ed and received subcutaneous implantation
of silastic tubes containing testosterone,
and then behavioral tests were carried out.
Next, the STC males received bilateral
medial amygdala lesion (AMGL) followed by
further behavioral tests.
In the tests after STC, although most
of STC males showed male sexual behavior,
mount and intromission frequencies were
lower than those of sham-operated males.
Ejaculation was eliminated after STC. On
the tests after the second brain surgery,
further decrease of copulatory activity was
observed in males with both STC and AMGL.
The results indicate a possibility of the
pathway, other than the stria terminalis,
of the amygdala in neural control of
copulatory behavior in the rat.
Tokyo, Japan.
Avian reproductive endocrinology has been mostly
studied using poultry species such as chickens and turkeys
or small passerine species in laboratories or in field. To
understand mechanisms in avian seasonal breeding in each
species, studies in different groups of birds which have
different adaptational strategies are must. Fortunately we
have a chance to collect blood samples from wild
wintering population of pintails at Sin-hama duck-netting
preserve owned by the Imperial Household. Samples were
collected from male and female pintails once a month from
November 1991 through January 1992 and twice in
February 1992. Behavioral observation was carried out at
Gyotoku Waterfowl preserve and Ueno Shinobazu Pond.
Plasma concentration of luteinizing hormone (LH) in both
sexes were low in November and December. Plasma
concentrations of LH in males began to increase in January
and became more than 1 ng/ml on February 12, but those
in female were still low even on February 12. Male
pintails showed courtship behavior in February but females
did not respond it.
HORMONAL INDUCTION OF COURTSHIP BEHAVIOR
IN THE NEWT, CYNOPS PYRRHOGASTER.
F.Toyoda', S.Tanaka? and S.Kikuyama? .
'Dept. of Physiol., Nara Med. Univ.,
Kashihara, *Inst. of Endocrinol., Gunma
Unv., Maebashi, “Dept. of Biol. Sch. of
Educ., Waseda Univ., Tokyo.
In breeding season, the sexually matured
male newt vibrates the tail in front of
the female at an early stage of courtship.
The vibration- behavior is elicited in
sexually inert males by injection of
prolactin (PRL) and/or gonadotropin (GTH) .
This effect of GTH was nullified by
castration. In the castrated Male
receiving PRL, both testosterone
propionate (TP) and dihydrotestosterone
(DHT) were effective in eliciting the
behavior, whereas estradiol was not
effective. The male-like behavior was also
observed in a considerable number of
ovariectomized females with low
frequencies. In these females,
administration of PRL together with TP,
but not with estradiol, was effective in
increasing the frequency of the male-like
behavior. The incidence and frequency were
not affected by the difference in the sex
of the partner.
1256 Endocrinology
RAINBOW TROUT 3B-HYDROXYSTEROID DEHYDROGENASE
/A54-ISOMERASE: cDNA CLONING AND ENZYMATIC
PROPERTIES
N. Sakai*, M. Takahashi, M. Tanaka and Y. Nagahama
Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki 444,
*Dept of Marine Biosci., Fac. of Biotech., Fukui Pref. Univ.,
Matsuoka 910-10
3B-Hydroxysteroid dehydrogenase/A5+-isomerase (3B-
HSD) is the enzyme in the steroidogenic pathway controlling
the formation of A‘-3-ketosteroids from A5-3B-
hydroxysteroids. A cDNA clone encoding 3B-HSD was
isolated from a cDNA library of rainbow trout (Oncorhynchus
mykiss) ovarian thecal cell layers. The cDNA contained an
open reading frame of 1122 nucleotides encoding a protein of
374 amino acid residues. The deduced protein has 48%
homology with 3B-HSD of human and bovine, 45%
homology with that of mouse and 3 B-HSD type II of rat, and
44% homology with 3B-HSD type I of rat. Expression of
trout 3B-HSD cDNA in nonsteroidogenic mammalian COS-1
cells led to production of an enzyme which is capable of
converting pregnenolone, 17@-hydroxypregnenolone and
dehydroepiandrosterone to progesterone, 17 a-hydroxy-
progesterone and androstenedione, respectively. The
conversion of three A5-3B-hydroxysteroid substrates to A+-3-
ketosteroids by the activity of trout 3B-HSD expressed in
COS-1 cells are almost the same. The cDNA hybridized to a
single species of mRNA (1.5-kb in length) in rainbow trout
ovary RNA. The 1.5-kb transcript markedly increased in
trout ovaries during oocyte maturation.
RAINBOW TROUT CHOLESTEROL SIDE-CHAIN CLEAVAGE
CYTOCHROME P450 ( P450scc ): cDNA CLONING AND
mRNA EXPRESSION
M. Takahashi, M.Tanaka, N.Sakai, S.Adachi and Y.Nagahama,
Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki 444
P450scc catalyzes the conversion of cholesterol to
pregnenolone, the initial rate-limiting reaction in the
synthesis of several steroid hormones. A cDNA clone
encoding P450scc was isolated from a rainbow trout
( Oncorhynchus mykiss ) ovarian thecal cell layer cDNA
library. The cDNA contains an open reading frame of 1,542
nucleotides encoding 514 amino acid residues. The deduced
amino acid sequence of rainbow trout P450scec shows 48%
homology with that of human, and 46% homology with that
of rat, bovine and pig. P450scc activity was confirmed by
transfected COS-1 monkey kidney tumor cells containing
P450scc cDNA and subsequent detection of conversion of 25-
hydroxycholesterol to pregnenolone by radioimmunoassay.
Northern blot analysis revealed a single species of mRNA
approximately 1.8 kb in length. The RNA transcripts were
not detected in early vitellogenic follicles but were present in
postvitellogenic follicles and were abundant in postovulatory
follicles.
RNA EXPRESSION OF SEROIDOGENIC ENZYMES BY
RAINBOW TROUT OVARIAN FOLLICLES JIN VITRO
M.Tanaka, M.Takahashi, N. Sakai and Y. Nagahama. Lab.
of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki 444
Postvitellogenic follicles of salmonid fishes produce
predominantly 170,20B-dihydroxy-4-pregnen-3-one (17a,
20B-DP, the maturation-inducing hormone) in response to
gonadotropin. Forskolin can mimic this action of gonado-
tropin. Expression of mRNAs for four ovarian steroidogenic
enzymes (P450scc, 3B-HSD, P450c17 and P450arom)
involved in oocyte maturation was investigated in ovarian
follicle layers incubated in the presence or absence of
forskolin. The level of P450scc mRNA remained constant
during the first 24 hr and then increased at 36 hr in the
presence of forskolin, coinciding with an incease in
17a,20B-DP production. 3B-HSD mRNA increased within
24 hr of incubation with forskolin, and then retumed to the
initial level at 36 hr. mRNAs for P450c17 and P450arom
decreased rapidly regardless of forskolin. Actin mRNA
remained constant within 24 hr of incubation. Dynamic
changes of mRNAs may constitute part of the mechanism
underlying the increased capacity of gonadotropin-
stimulated ovarian follicles to produce 17 a,20B-DP.
GONADOTROPIN-INDUCED STEROID PRODUCTION IN
TESTICULAR TISSUE OF THE JAPANESE EEL, ANGUILLA
JAPONICA
T. Kobayashi!, T. Miura!, K. Yamauchi2, N. Sakai3, M.
Tanaka! and Y. Nagahama!. 'Natl. Inst. for Basic Biol.,
Okazaki 444, 2Hokkaido Univ. and 3Fukui Pref. Univ.
In the cultivated Japanese eel, human chononic
gonadotropin (HCG) induces spermatogenesis by stimulating
the testicular production of 11-ketotestosterone (11-KT).
The effect of HCG treatment on the pattern and yield of
steroid metabolites were examined using a cell-free system.
Cell-free homogenates of eel testes before and 24 hours after
HCG treatment were incubated for 1 hour with 'C-labeled
steroids and the metabolites were analyzed by TLC.
Analysis of steroid biosynthetic pathways showed that the
testicular homogenates synthesized 11-KT via the *A-pathway
(pregnenolone — 17a-hydroxypregnenolone — dehydroepi-
androsterone). When !4C-pregnenolone and 'C-dehydro-
epiandrosterone were used as substrates, a significant increase
in androstenedione production was observed in testicular
homogenates obtained after treatment. In incubations with
14C-testosterone, androstenedione and 11-KT were detected
as the major ; however, there was no difference in the pattern
and yield of metabolites between control and HCG-treated
groups. These results indicate that HCG increases 11-KT
production by stimulating the activity of 3 B-hydroxysteroid
dehydrogenase/AS+-isomerase, thereby accelerating the
conversion of dehydroepiandrosterone into androstenedione.
Endocrinology 1257
ACTIVATION OF SOMATOLACTIN CELLS IN THE
PITUITARY OF THE RAINBOW TROUT BY LOW
CALCIUM ENVIRONMENT.
S.Kakizawa, T.Kaneko, S.Hasegawa, T.Hirano
and H.Kawauchi*. Ocean Res. Inst., Univ.
of Tokyo, Tokyo, *School of Fisheries
Sciences, Kitasato Univ., Sanriku.
Somatolactin (SL) is a putative
pituitary hormone, structurally related to
both prolactin and growth hormone. To
clarify the possible involvement of SL in
calcium regulation, we examined the
effects of ambient calcium on activity of
SL cells in the rainbow trout.
The fish were transferred from fresh
water (FW) to FW containing 10mM CaCl
(Ca-FW) or to 80% sea water (SW) and from
Ca-FW to FW. The pituitary was fixed 10
and 21 days after transfers. The
pituitary sections were immunocyto-
chemically stained with anti-chum salmon
SL serum. On the adjacent section, the
presence of SL mRNA was examined by in
situ hybridization using a cDNA probe
encoding chum salmon SL. Both cellular
and nuclear cross-sectional areas were
decreased when fish were transferred from
FW to Ca-FW or to SW; no change was seen
after transfer from FW to FW. The level
of SL-mRNA decresed 10 days after transfer
to Ca-FW. On the other hand, not only
cellular and nuclear areas but also SL-
MRNA levels increased after transfer from
Ca-FW to FW. The results support a
hypercalcemic role for SL.
IMMUNOCYTOCHEMICAL INVESTIGATIONS OF
GTH CELLS IN THE PITUITARY OF NILE
TILAPIA (OREOCHROMIS NILOTICUS) DURING
THE EMBRYONIC DEVELOPMENT.
T.Saga !, S. Adachi 1, K. Yamauchi !, H. Kagawa 2, H.Tanaka2
and H.Takahashi!, Faculty of Fisheries, Hokkaido University.
Hakodate, 2. National Research Institute of Aquaculture, Mie.
In the pituitary of adult tilapia, glandular cells
immunoreactive to anti-Pagrus major GTH Ip
antiserum (GTH-ir cells) were localized mainly in the
ventral part of proximal pars distalis (PPD) and
additionally in rostral pars distalis (RPD) and in the
peripheral region of pars intermedia (PI). The cells
distributed in the PPD had large vacuoles in the
cytoplasm, while most of the cells distributed in the
peripheral PI had no vacuoles. Both small granules
and large globules in the GTH-ir cells revealed
immunoreactivity.
During embryonic development, the pituitary first
occurred at the ventral part of the third ventricle 72 hr
after fertilization. In this stage, a few GTH-ir cells
were detected in the centro-dorsal part of the pituitary
just posterior to PRL-ir cells, but the GTH-ir cells
had no vacuoles. Eighty four hr after fertilization,
just before hatching, the pituitary developed further
and GTH-ir cells were distributed between the PRL-ir
and GH-ir cells. In this stage, the GTIlI-ir cells
increased in number, but vacuoles were not observed
in the cytoplasm. After hatching, the GTII-ir cells
shifted their position to the ventral part of PPD.
GTH-ir cells revealed a similar distribution pattern to
that seen in adult pituitary 50 days after fertilization,
but the cells distributed in the PPD and PI had no
vacuoles.
IMMUNOHISTOCHEMICAL LOCALIZATION OF MELANOCORTIN IN THE
BRAIN AND HYPOPHYSIS OF THE CLOUDY DOGFISH AND CHANGE
WITH ONTOGENETIC DEVELOPMENT
A.Chiba and Y.Honma. “Dept. of Biol., Sch. of Dent. at
Niigata, Nippon Dent. Univ., Niigata,*Sado Mar. Biol.
Stat., Fac. of Sci., Niigata Univ., Niigata.
Using specific antisera raised against a-melanocyte
stimulating hormone(a-MSH) and adrencorticotrophic
hormone (ACTH), we examined the localization of immuno-
reactivity in the brain and hypophysis of the cloudy
dogfish, Scyliorhinus torazame, and its change with
ontogenetic development. For the purpose, 6 adults, 2
juveniles and 3 embryos were studied. In the adults,
melanocortin (a@-MSH and ACTH)-like immunoreactivities
were demonstrated in the brain and two adenohypophysial
lobes (the pars distalis and the pars intermedia) with
some topographic variations in the brain: the a-MSH-
immunoreactive perikarya were found in the nucleus
tuberculi posterioris, the n. sacci vasculosi, the n.
lateralis tuberis, the n. medius hypothalamicus and the
posterior recess organ, whereas the ACTH-immunoreactive
cell bodies were localized in the n. lateralis tuberis
and some parts of the posterior recess organ. Immuno-
reactivity was entirely absent in the embryos (15-35mm
in total length) examined. In 1- and 50-day-old juveniles,
melanocortin-immunoreactive structures similar to those
of adults were demonstrated, although they were less
dense than in the adults. These findings suggest
multiple function of melanocortin as hormone and neuro-
regulator in the dogfish hypothalamo-hypophysial system
INFLUENCE OF ENVIRONMENTAL SALINITY ON
CHLORIDE, PROLACTIN AND GROWTH HORMONE
CELLS IN OREOCHROMIS MOSSAMBICUS YOLK-SAC
LARVAE
F.G. Ayson, T. Kaneko, S. Hasegawa and T.
Hirano, Ocean Research Institute,
University of Tokyo, Nakano, Tokyo 164
Newly-hatched larvae of OF,
mossambicus survive direct transfer from
FW to SW (33 ppt); larvae 2 days old or
older can tolerate direct transfer from FW
to 25 ppt SW. Because the major adult
osmoregulatory organs are still poorly
developed in early larval stages, chloride
cells (CC) in the yolk-sac membrane were
sought, using DASPEI, a stain specific for
mitochondrion-rich cells. Changes in CC
size and density through complete yolk
resorption were recorded following
transfer of newly-hatched larvae from FEW
to SW. Prolactin (PRL) and growth hormone
(GH) cells in the pituitary of the larvae
were also examined by immunocytochemistry.
Following transfer to sw, cc size
increased significantly to 2- to 4-fold
over that seen in FW larvae. The mean
size of PRL cells and the area of the
pituitary containing PRL cells were
significantly larger in FW larvae than in
SW larvae. GH cell size was not affected
by transfer of larvae to SW. These results
suggest the possible involvement of CC in
the yolk-sac membrane and of pituitary PRL
cells in osmoregulation of O. mossambicus
yolk-sac larvae.
1258 Endocrinology
COLD ADAPTATION AND PITUITARY OF THE SAFF-
RON COD, ELEGINUS GRACILIS.
M. Ogawa
Dept.Biol.,Saitama Univ.,Urawa,Saitama.
Most of the Antarctic teleosts has been
reported to have an aglomerular kidney and
the antifreeze glycoprotein in their sera.
Saffron cod survives the ice-laden coastal
waters during the winter by possessing hi-
gh concentration of antifreeze glycoprote-—
in in the serum(Burchman et al.,1984).
The seasonal variations of plasma osmo-
lality, kidney structure and pituitary of
the saffron cod were investigated.
Plasma osmolality was increased during
the winter than during the summer. In the
winter fish, atrophy of glomerulus was ob-
served. This may be related to the conser-
vation of antifreeze glycoproteins for
cold water. Growth hormone(GH)- and prola-
ctin(PRL)-secreting cells in the pituitary
were identified by immunohistochemical
staining used antisera raised against the
chum salmon hormones. In the summer fish,
the positive immunoreactions with both GH
and PRL were remarkable than those of the
winter fish.
Recently it is known that GH blocks the
antifreeze glycoprotein mRNA transcription
in the liver. However, these results may
suggest the possibility of PRL clearing of
antifreeze glycoprotein of the serum by
increasing glomerular filtration during
summer.
PROLACTIN ENHANCES CELL PROLIFERATION OF
EXOCRINE PANCREAS IN MICE
M.Matsuda, T.Mori, M.K.Park and S.Kawashima
Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo
Prolactin (PRL) has a diverse action on
various tissues in vertebrates. Recently,
PRL is known to stimulate proliferation of
rat pancreatic B-cells, although no report
is available for PRL effects on the
exocrine Pancreas. However, we have
demonstrated that in mice pituitary
isografting increases pancreatic weight,
associated with hyperprolactinemia. The
weight increase is supposed to be due to
the proliferation and/or hypertrophy of
pancreatic acinar cells, since B-cells
constitute only a small part of the total
pancreatic volume.
In this study, an anterior pituitary
gland was grafted under the renal capsule
in 6-wk-old male SHN mice (PG mice).
Pituitary grafting resulted in marked
increases in the weight and DNA content of
the pancreas in PG mice at 30 days compared
to those in controls bearing no graft.
However, DNA and protein contents per
tissue weight of the pancreas were less
than controls, suggesting that hypertrophy
of acinar cells also contributes to the
weight increase of pancreas. Rate of DNA
synthesis determined by BrdU labeling was
greater in both endocrine and exocrine pan-
creas in PG mice than in controls. There-
fore, pituitary grafting stimulates the
proliferation and hypertrophy of pancreatic
acinar cells as well as islet cells.
PROLIFERATION OF ANTERIOR PITUITARY CELLS
IN STREPTOZOTOCIN-INDUCED DIABETIC RATS.
S.Takahashi. Dept.of Biol., Fac. of Sci.,
Okayama Univ., Okayama.
Insulin is involved in the cell
proliferation of fibroblasts, hepatocytes
and some of cell lines. The purpose of
the present study is to clarify the role
of insulin in the proliferation of rat
Pituitary cells. Male rats of the
Wistar/Tw strain were intraperitoneally
Ziven streptozotocin (STZ) at a dose of
32.5 mg/kg. STZ-treated rats showed high
blood glucose levels and low serum insu-
lin levels. Cell proliferation was exam-
ined by the detection of bromodeoxyuri-
dine (BrdU)-uptake cells. Estradiol-17B8
(504g) was given to stimulate the pro-
liferation of pituitary cells.
The number of BrdU-uptake cells was
lower in STZ-treated rats than in
controls. The responsiveness to estrogen
on the cell proliferation was lower in
STZ-treated rats. Insulin treatment in
STZ-treated rats increased the number of
BrdU-uptake cells and recovered the
responsiveness to estrogen. Particularly,
insulin stimulated the proliferation of
prolactin cells in estrogen-treated rats.
These results suggest that insulin is
necessary to the proliferation of pitui-
tary cells.
SUPPRESSION OF THE DEVELOPMENT OF UTERINE
ADENOMYOSIS BY DANAZOL TREATMENT IN MICE
T. Singtripop?, T. Mori!, S. Sakamoto?
S. Sassa*, M.K. Park+ and S. Kawashima?+
+Zool. Inst., Fac. Sci., Univ. Tokyo and
2Dept. Endocr., Med. Res. Inst., Tokyo
Med. Den. Univ., Bunkyo-Ku, Tokyo 113
Danazol, a Synthetic gonadotropin inhibi-
tor, has been used clinically in the treat—
ment of uterine adenomyosis and various
breast disorders. In this study, the inhi-
bitory effects of danazol on the sponta-
neously developed- and experimentally in-
duced-adenomyosis were studied in SHN mice.
Female mice were given daily injections
of 0.5 ug danazol for 5 weeks during 4-9 or
9-14 weeks of age. To accelerate the gene-
sis of adenomyosis, the other group of mice
was ‘isografted with anterior hypophysis in-
to the uterine lumen at 5 weeks of age.
A half of this group was given danazol for
4-9 weeks of age. Mice receiving sesame oil
only served as controls. All mice were
killed at 21 weeks of age.
The results showed that danazol treatment
resulted in a Significantly lower incidence
of Spontaneous development of adenomyosis
than the control, and also inhibited the
genesis of experimentally induced-adenomy-
osis. Furthermore, danazol treatment
caused the decrease serum levels of LH and
PRL associated with hypofunction of ovaries
and persistent diestrus. These results sup-
port the usefulness of danazol for the cli-
nical treatment of gynecological disorders
except for hypofunction of ovaries.
Endocrinology 1259
MORPHOLOGICAL STUDY OF MOUSE _ PITUITARY
CELLS CULTURED ON COLLAGEN- OR _ POLY-L-
LYSINE-COATED_ SUBSTRATA. J
K.Nakatomi, S.Takahashi_ and_Y.Kobayashi,
Dept.of Biol., Fac.of Sci., Okayama Univ.,
Okayama.
In vitro stud S_necessar to stud
an
u ay
mechanism | of Bs differentiation
proliferetion of anterior pituitary cells.
The purpose of this study was to clarify
the effects of culture substrata_ on
dissociated mouse pituitary cells. Type
PeeyeS collagen-, or _ poly-l-lysine-
coated PLL) glass coverslips, an non-
coated glass ‘coverslips (control) were
used as culture substrata.
The anterior Gey cells from male
ICR mice (5-s weeks of age were
dissociated with 0.5% trypsin at ine for
15 min. Pituitary cells were suspended in
Dulbecco modifie Eagles' medium
containin 20mM HEPES supplemented with
10% feta calf serum. he dissociated
cells (2.2x 10° cells/well) were seeded on
‘Gey Ea ae coat edit En Oy it : 4
ug/cm2 o pe or type collagen an
O, . (0) EB £ PLL. Growth
hormone {GH) ‘and prolactin (PRL) cells
were immunocytochemiclly detected at 3 and
5 days of culture. We found two Eypes of
GH and PRL cell, round-shaped cells and
elongated cells. The relative propotions
of round-shaped GH or PRL cells changed
with the concentrations’ of eee ue fv
collagen or PLL. The number of and PRL
cells was highest in the culture condition
of type I collagen-coated substrata
(a ney eee This result indicats that SE
I collagen is the most suitable
substratum. Under this culture condition,
effect of GH-releasing factor (GHRH on
the mumber of GH _ cells was_ studied.
PSE °M) treatment for 48hr PETE a
1.3-fold increase in the percentage of GH
compared with controls. These
suggest that meno Toay: of
pituitary cells is affected
z py culture substrata (ex. collagens
Ss).
cells
results
cultured
artl
ss PE
EFFECT OF NEUROPEPTIDES ON CHANGES’ IN
MAMMOTROPH NUMBER
-FLOW CYTOMETRIC ANALYSIS-
T. Shinkai and H. Ooka. Dept. of Cell
Biology, Tokyo Metropol. Inst.of Gerontol.
Tokyo.
We developed a procedure for detection
of pituitary mammotrophs with flow
cytometry and analyzed the effects of
neuropeptides on changes in the mammotroph
number in vitro using the technique. Rat
anterior pituitary cells, were cultured in
24-well plates at 8x10°*/well with 1 ml
medium. After fixation of the cells with
Bouin's solution, they were treated by the
flow-cytometry technique. The number of
mammotrophs increased in basal culture
medium. The addition of GRF suppressed the
increase in the mammotroph number. The
addition of LHRH accelerated the increase
in it. These results were similar to those
of light microscopic examination. Other
neuropeptides(PRL releasing peptides: PHI,
substance P, angiotensin II, B-endorphin,
leucine enkephalin, TRH, vasopressin, VIP,
oxytocin and galanin, other peptides:
bradykinin and NPY) increased in the
mammotroph number up to the control level
and had no effect on the changes in the
mMammotroph number. These results indicate
that PRL releasing factors are not always
proliferation factors for the mammotroph.
,
PHOSPHORYLATION OF THE PIT-1
TRANSCRIPTION FACTOR IS NOT ESSENTIAL TO
PROLACTIN GENE ACTIVATION IN MtT/S CELLS
S. Nagata’ and Michael G. Rosenfeld2. 1Dept.
Material and Biol. Sci., Fac. Sci., Japan Women’s
Univ., Tokyo and Howard Hughes Med. Inst., School of
Med., Univ. of California, San Diego.
Growth-hormone (GH)-producing MtT/S cells
switch to prolactin (PRL) production upon cultivation
with estrogen and IGF-1. Western immunoblot
analyses of the nuclear and cytoplasmic extracts
showed no change in the Pit-1 contents in PRL gene
activation. Incorporation of [85S]-amino acids into Pit-1
and nuclear translocation of the labeled Pit-1 were
slightly stimulated as detected by SDS-PAGE analyses
of the immunoprecipitates . After incubation of the cells
for 3 hr with inorganic 32P, [32P]-Pit-1 was
immunoprecipitated from the cytoplasmic extract.
However, trace amount of [82P]-Pit-1 was detected in
the nuclear extract. Stimulation of the cells with
estrogen and IGF-1 for 20 min, 6 hr, 96 hr did not alter
the amount of [82P]-Pit-1. EGF stimulated Pit-1
phosphorylation by itself and enhanced estrogen/IGF-1-
induced PRL gene activation. But EGF alone could not
induce PRL. Therefore, availability of phosphorylated
or non-phosphorylated Pit-1 may not be essential for
estrogen/IGF-1-induced PRL gene activation in MtT/S
cells. Involvement of additional factors interacting Pit-
1 should be considered.
INVOLVEMENT OF MAMMALIAN GROWTH
HORMONE AND PROLACTIN IN INDUCTION
OF VITELLOGENIN IN THE PRIMARY
CULTURE OF EEL HEPATOCYTES.
H.-C. Kwon, Y. Mugiya. Lab. Physiol., Fac.
Fish., Hokkaido Univ., Hakodate.
The role of pituitary hormones in vitellogenin
(Vg) synthesis was investigated using the primary
culture of immature eel hepatocytes in serum free
medium. When cultured with estradiol-178 (E2,
2x10 6M) alone, little or no Vg was found in the
medium throughout an experimental period of 9
days. Estradiol-178 administration to hypophy-
sectomized eels also failed to induce in vivo Vg
synthesis. However, cultures added E2 together
with mammalian growth hormone (GH) or
prolactin (PRL) induced Vg synthesis and the rate
of its production gradually increased untill at least
9 days. Furthermore, even in the culture of
hepatocytes obtained from immature eels primed
from E2 administration, the addition of E2 alone did
not show the increased synthesis of Vg, while the
combination of E2 and GH or PRL stimulated Vg
synthesis. Nither GH alone or PRL alone had any
effect on Vg induction. These results demonstrate
that the pituitary of eel may be involved in the
hepatic synthesis of Vg.
1260 Endocrinology
THE EFFECT OF DIFFERENT REGIONS OF BRAIN TISSUE
ON CELL PROLIFERATION OF THE ADENOHYPOPHYSIAL
PRIMORDIUM
M. Shirai and Y. G. Watanabe
Dept. of Biol., Fac. of Sci., Niigata Univ., Niigata.
We previously reported that in fetal rats the
developing diencephalic floor stimulated prolifera-
tion of adenohypophysial primordial cells. In this
study, we investigated if other parts of brain
increase the rate of cell proliferation in the
adenohypophysial primordium by the use of the BrDU
method. For this purpose, the rostral end of the
telencephalon and parts of the diencephalon ( its
roof, posterior wall and chiasmal area ) were
selected and their effect on the developing adeno-
hypophysis was compared with the effect of the
diencephalic floor. The effect of surrounding
mesenchymal tissue was also studied
The adenohypophysial primordia were removed from
rat fetuses on day 13.5 of gestation and cultiveted
with several different regions of brain. Other than
the diencephalic floor facing the adenohypophysis
the posterior wall of the diencephalon ( the
presumptive mammillary body ) augmented the prolife-
rative rate of adenohypophysial cells. Mesenchymal
tissue had no effect on cell proliferation.
These results suggest that the developing dien-
cephalic floor specifically contains some factor(s)
which is essential for the proliferation of adeno-
hypophysial primordial cells.
WHAT AN ANOMALY OF THE FETAL RAT TELLS ABOUT THE
DEVELOPING BRAIN-ADENOHYPOPHYS!AL RALATIONSHIP
Y. G. Watatabe and H. Haraguchi
Dept. of Biol., Fac. of Sci., Niigata Univ., Niigata
We have hitherto presented in vitro data showing
that the developing diencephalic floor is involved
in growth and cytogenesis of the rat adenohypophysis.
In the course of a study on the adenohypophysis we
have found an abnormal rat fetus in which adeno-
hypophysis was out of contact with the brain. Since
experiments of brain-adenohypophysis disconnection
are generally difficult in mammalian fetuses, histo-
logical study of such adenohypophysis is of quite
interest. The results are summarized as follows:
1) The volume of this abnormal adenohypophysis
was far smaller ( less than 1/10 ) than that of the
normal gland. 2) When immunostained with anti- aMSH
serum, the definitive pars intermedia was not ob-
served. 3) A small number of immunoreactive cells
were seen and after staining with antisera against
ACTH, GH, TSHB and LHB. Whereas PRL cells could
not be found
All of these data suggest an important role of
the diencephalic floor in the normal histogenesis of
the adenohypophysis especially of the intermediate
lobe.
FINE STRUCTURAL STUDY ON THE PITULTARY—ADRENAL SYSTEM OF
MICE UNDER PROLONGED EXCESS LIQUID INTAKE.
S.Thara, S.Nishibayashi, S.Takeuchi, S. Takahashi and Y.
Kobayashi. Dept. of Biol., Fac. of Sci., Okayama Univ.
Okayama.
It has been previously demonstrated that induced
polydipsia(ca. 200% v/w/day) by giving a 5% glucose
solution concomitant with food deprivation resulted in
marked hypersecretion in cells of the mouse pituitary
intermediate lobe. Despite malnutritional condition of
this excess drinking animals can survive for 3 weeks or
so.
In the present study the effects of prolonged liquid
intake on the pituitary and adreal glands were
investigated. Animals were divided into 3 groups, (1)
normal controls, (2) mice given a 4% powedered milk
solution without food for 3 weeks and (3) mice given a.
5% glucose solution alone for 3 weeks. Their body
weights increased to 115% of the initial value in group
1 but decreased to 70.9% and 62.4% in groups 2 and 3,
respectively, both of which showed a marked increase in
liquid consumption during the time of experiment.
Morphometrical analysis of the pituitary intermediate
lobe cells at the electron microscopic level indicated
hypersecretion in group 3. Cell atrophy and involution
of the pituitary anterior lobe were recognized. The wet
weight of organs/g body weight decreased in those
including the pituitary, thymus, spleen, and kidney, and
no change in pancreas and testis. The adrenal glands
showed weight gain/g body weight. These results
indicated that prolonged liquid intake, especially in
mice of 5% glucose drinking, stimulated the secretory
activity of the pituitary intermediate lobe cells that
due probably not to malnutrition but to polydipsia.
DIFFERENTIAL DISTRIBUTION OF BIOACTIVE
PEPTIDES RELATED WITH DRINKING BEHAVIOR IN
THE RAT SUBFORNICAL ORGAN. =
T. Yamamoto, T. Hirohama, S. Nakamura, and
H. Uemura. Biol. Lab., Kanagawa Dent.
Coll., Yokosuka.
The distribution o loactive peptides
related with drinking behavior, namely
substance P (SP), Leu-enkephalin (Leu-
enk), neurotensin (NT), cholecystokinin
(30-39, CCK), LHRH, vasopressin (VP), and
B-endorphin, was immunohistochemically
studied on the rat subfornical organ
(SFO), one of the drinking center in the
central nervous system. Leu-enk- and SP-
immunoreactive fine fibers were located on
the anterodorsal half of the SFO. On the
other hand, NT- and CCK-immunoreactive
fibers were observed in the central
portion of the SFO. Although a few thick
LHRH-immunoreactive fibers were seen in
the central portion of the SFO, more
fibers were aligned on the ventral midline
of the fornix where glial elements were
condensed. A few very fine VP-
immunoreactive fibers were also detected
Mainly on the ependymal tissue covering
ventral surface of the SFO. Endorphin-
immunoreactivity was not detected in the
SFO although extensive immunoreactive
fibers were observed, for example, in the
hypothalamus.
These results suggest the differential
termination of peptidergic fibers in the
SFO and ecueeeenl differences within the
SFO in contribution of bioactive peptides
to drinking behavior.
Endocrinology 1261
PRODUCTION AND CHARACTERIZATION OF
SPECIFIC ANTIBODY AGAINST a-SUBUNIT OF
RAT PITUITARY GLYCOPROTEIN HORMONES
S. Tanaka, H. Mochida, S. Kurabuchi!, H. Hayashi, K.
Wakabayashi and K. Kurosumi (Inst. of Endocrinol., Gunma
Uniy., Maebashi and INihon Dental Univ., Tokyo)
To obtain an antibody specific for a-subunit of rat pituitary
glycoprotein hormones, we synthesized a peptide
corresponding to the sequence 37-53 (Phe-Ser-Arg-Ala-Tyr-
Pro-Thr-Pro-Ala-Arg-Ser-Lys-Lys-Thr-Met-Leu-Val) of the
rat @-subunit. The polyclonal antibody against this peptide
was generated in rabbit. This region is hydrophilic, and
highly conservative among several mammalian species. This
antiserum immunostained two types of cells in rat anterior
pituitary: the one type contained LHB, and the other, also
TSH. The anterior pituitaries of mouse, ovine, and porcine,
which have identical sequence of the present peptide were
immunostainable with this antiserum. However, human (45-
Leu in a-subunit) and bullfrog (45-Met) were not stained
with this antiserum. Similarly, using this antiserum the
immunoreactive cells were found only in the anterior
pituitaries of Urodele, Rhacophorus, and Hyla groups among
fifteen amphibian species including Rana group examined in
this study. These data suggest that this antibody is
sequence specific and useful to immunological study.
HEMORRHAGE AFFECTS THE ENDOTHELIN CONTENTS
IN THE RAT PITUITARY.
Se Nakamura!, 1, Yamamoto!, M. Naruse2, K.
Naruse2, K.Aoki3, T.Hirohama', H.Uemura'.
1TBiol. Lab., Kanagawa Dent. Coll., Yoko-
suka, 2Dept. of Med., Tokyo Women's Med.
Coll., Tokyo, 3Div. of Biol., Natl. Inst.
Radiol. Sci., Chiba.
To investigate the role of endothelin
(ET) in the pituitary, ET-1 and ET-3 were
measured by enzyme immunoassay in rats
subjected to hemorrhage via the cannula
inserted into the external carotid vein.
Plasma levels of arginine vasopressin
(AVP) were also estimated by radioimmuno-
assay.
Hemorrhage (4 ml/260-275 g body weight)
induced a significant elevation of plasma
AVP levels. The content of ET-1 in the
neural lobe [155 + 65 (n=7) vs 414 + 35
(n=9) pg/g wet weight], but not that of
ET-3 [456 + 112 (n=6) vs 311 + 68 (n=5)
pg/g], significantly increased immediately
after hemorrhage. In the glandular lobe,
hemorrhage significantly reduced both ET-1
[88.4 + 13.4 (n=8) vs 41.1 + 2.7 (n=10)
pg/g, Aspin-Welch method] and ET-3 [731 +
95 (n=8) vs 361 + 66 (n=9) pg/g] levels.
The present results suggest that ET-1
and ET-3 in the pituitary are involved in
mediating the physiological responses to
hemorrhage in the rats.
APPLICATION OF A REVERSE HEMOLYTIC PLAQUE
ASSAY FOR DETECTION OF HORMONE RELEASE FROM
AMPHIBIAN PROLACTIN CELLS
T.Kawasaki,K.Nakajima,T.Kouki and
S.Kikuyama
Dept. of Biol., Sch. of Educ., Waseda
Univ., Tokyo.
Reverse hemolytic plaque assay was in-
troduced to study the response of in-
dividual prolactin cells in a mixed culture
of bullfrog(Rana catesbeiana) distal lobe
cells to TRH(pyro Glu-His-Pro NHz2). The
procedures were essentially the same as
those described by Frawley et al.(1983).
In the present experiment, medium 199
diluted to 75% was used for culture. Anti-
prolactin antiserum used in the plaque as-
say and for the immunohistochemical detec-
tion of prolactin cells was produced in a
rabbit immunized with highly purified
bullfrog prolactin. Both rate of plaque
formation and mean plaque area were in-
creased according to the time of TRH(10~°M)
treatment over the period of 90 min, and
also in response to the concentrations of
TRH(107%-107-7°M) as measured after 90 min
of treatment.
MOLECULAR CLONING AND NUCLEOTIDE SEQUENCE
ANALYSIS OF cDNA FOR TOAD PROLACTIN
N.Takahashi, K.Yamamoto, and S.Kikuyama.
Dept. of Biology, Sch. of Education,
Waseda Univ.
The cDNA encoding C'-terminal sequence
of toad (Bufo japonicus) prolactin (PRL)
was specifically amplified employing
polymerase chain reaction (PCR) method
from cDNA encoding mRNA extracted from
anterior pituitaries. Sequence analysis
revealed that this clone contains 602bp
nucleotides and is encoding 134 amino
acids of C'-terminal region of toad PRL
molecule. This region of toad PRL had
Qiks, SSB, Gils, GO%w, GOB Amel Wey
homologies with the corresponding region
of bullfrog, salmon, sea turtle, chicken,
porcine and human PRLs. Northern blot
analysis carried out using this cDNA as a
probe revealed that PRL mRNA consists of
about 1.0 kb nucleotides.
1262 Endocrinology
ELEVATION OF PROLACTIN mRNA LEVELS BY NA-
TIVE AND SYNTHETIC TRHs IN THE BULLFROG
DISTAL LOBE.
S.Sasaki, N.Takahashi, K.Nakajima and
S.Kikuyama.
Dept. of Biol., Sch. of Educ., Waseda
Univ., Tokyo.
We have obtained a substance which has
prolactin-releasing activity from the
bullfrog hypothalami. This substance was
identified as TRH (pyroGlu-His-ProNH2) by
means of radioimmunoassay, amino acid
analysis and reverse-phase high perfor-
mance liquid chromatography. Northern
blot analysis of prolactin mRNA in the
pituitary incubated in the presence of
the native or synthetic TRH(10~°M) were
performed using bullfrog prolactin cDNA
as a probe. AS a result, both samples
similarly elevated the prolactin mRNA
levels 2.5 times as high as the control
value with a concomitant rise of prolac-
tin levels in the medium, when measured
16h after incubation.
Two forms of prolactin exist in the
Xenopus distal lobe
K. YAMASHITA*, K. MATSUDA*, K. YAMAMOTO>,
Y. HANAOKA*, H. HAYASHI*, S. TANAKA* and
T. KATO®, S. KIKUYAMA*
*Dept. of Biol., Sch. of Educ., Waseda
Univ., Tokyo 169-50, #Inst. of En-
docrinol., Gunma Univ., Maebashi 371 and
*Kirin Co., Maebashi 371.
Two forms of prolactin (PRL), having
antimetamorphic activity as tested with
tail fragments of Xenopus larvae cultured
in the presence of Ta, were isolated from
the pituitary gland of Xenopus laevis by
extraction with acid acetone, and high
performance liquid chromatography (HPLC)
on anion exchange, gel filtration and
reverse-phase columns. The molecular
weight of both PRLS were 23,000 and
isoelectric points of xPRL I and II were
als eyeuel Bio esic Sequence homology between
one of the lysyl endopeptidase-digested
fragments from xPRL I and that from xPRL
II, which are comparable to bullfrog PRL
(116-172), was 88 %. XPRL I and II showed
73 and 68 % sequences homologies with
bullfrog PRL, respectively. They showed
little sequence homology with GH I and II.
EFFECT OF ARGININE VASOTOCIN (AVT) AND
AVT-RELATED PEPTIDES ON CONTRACTION OF IN-
TESTINE IN XENOPUS LAEVIS
D. Kuwagaki, S. Iwamuro, S. Kikuyama
Dept. of Biol., Sch. of Educt, Waseda
Univ., Tokyo
a
We have previously found that injections
of AVT to Xenopus induces defecation. This
lead us to study the effect of arginine
vasotocin (AVT) and AVT-related peptides
such as hydrin 1 (AVT-GKR) and hydrin 2
(AVT-G) on the contraction of Xenopus in-
testine in vitro. Xenopus intestine
(approximately 10 mm long) was immersed in
amphibian Ringers solution containing
various concentrations (10°°%-10°° M) of
the test substances. Changes of intestinal
length were measured by using a Magnus ap-
paratus. The intestine showed contraction
within 10 min in response to AVT, hydrin
1 and hydrin 2, their minimum effective
concentrations being 10°*, 10°” and 10°7 M
respectively. The contraction induced by
10-7 M AVT, hydrin 1 and hydrin 2 was
blocked by the simultaneous application of
10-@ M of [ d(CHz2)s,0-Me-Tyr*,Arg®]JAVP (V1
receptor antagonist) and [Pmp~*,0-Me-
Tyr7,Arg®]AVP (V1/V2 receptor antagonist),
but not of [d(CHz)s,D-Ile~,Ile*,Arg*]AVP
(V2 receptor antago-nist). The results in-
dicate that the effect of AVT and hydrins
on the intestinal contraction is mediated
through V1 type receptors.
EFFECT OF NEUROHYPOPHYSEAL SUBSTANCES ON
THE SECRETION OF IMMUNOREACTIVE JOINING
PEPTIDE BY THE BULLFROG DISTAL LOBE
S. Sugihara, S. lwamuro, and S. Kikuyama
Department of Biology, School of Education, Waseda University,
Shinjuku-ku, Tokyo 169-50.
In amphibians, joining peptide (JP) is processed from the multiple
precursor protein proopiomelanocortin (POMC) not only in the
intermediate lobe but also in the anterior lobe. Secretion of other
POMGC-derived peptides, such as ACTH and a-MSH, is known to be
affected by neurohypophyseal substances. Development of
radioimmunoassay (RIA) for JP enabled to ascertain whether the
neurohypophyseal substances also influence the release of
immunoreactive JP. Hemisected anterior lobes of adult bullfrog
pituitaries were incubated in 67% Eagle's MEM over the period of 3, 6,
12 and 24 h. Released JP in the medium was measured by RIA. The
release of immunoreactive JP increased according to the incubation
period and in response to AVT, hydrin2 (AVT-Gly), MT and TRH added
to the medium. In contrast, dopamine inhibited the release of
immunoreactive JP into the medium.
Endocrinology 1263
EFFECTS OF ARGININE VASOTOCIN AND PARA-
THYROID HORMONE ON CYCLIC AMP FORMATION
IN THE KIDNEY AND THE URINARY BLADDER OF
THE BULLFROG.
M. Uchiyama. Dept. Oral Physiol., School
Dentis. Niigata, Nippon Den. Univ.,
Hamaura-cho, Niigata.
Arginine vasotocin (AVT) and parathy-
roid hormone (PTH) are major regulators of
water and electrolyte excretion by the
kidney and the urinary bladder. In order
to elucidate the sites of actions of both
hormones in these organs, the hormone—
dependent cyclic AMP (cAMP) formation was
studied in vitro. The basal cAMP levels
are not significantly different among the
segments (proximal, distal and connecting
tubules and ureter) and the urinary blad-
der. AVT activated cAMP production in the
connecting tubules and the urinary blad-
der. Marked dose-dependent activation by
AVT was observed in preparations from the
connecting tubules and the urinary blad-
der. No activation by hPTH(1-34) was ob-
served in all preparations from the neph-
rons and the urinary bladder. These re-
sults suggest that Vo-type receptors are
present in the connecting tubules of neph-
ron and the urinary bladder. The lack of
hormonal activation of cAMP production ob-
served in some tissues may reflect either
the total absence or the presence in minor
quantity of hormonal receptors associated
with a cAMP-generating system in these or-
gans.
ISOLATION AND CHARACTERIZATION OF BULLFROG
THYROTROPIN
M. Sakai!, H. Hayashi, Y. Hanaoka2, S. Tanaka? and S. Kikuyama!
Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo.
2inst. of Endocrinol., Gunma Univ., Maebashi
Highly purified amphibian TSH was obtained for the first time from
the distal lobes of Rana catesbeiana. Glycoprotein fraction was
obtained from the acetone-dried pituitaries by ethanol precipitation.
This was subjected to hydrophobic interaction chromatography, anion
exchange chromatography, affinity chromatographies using anti-
bullfrog LHB and FSHB antibodies. Glycoprotein fraction thus obtained
had thyrotropic activity about 4 times as potent as bovine TSH and had
no LH (germinal vesicle breakdown-inducing) bioactivity. In addition, it
was confirmed that its thyrotropic activity was completely nullified by
the incubation with antibody against bullfrog LHa. This fraction was
subjected to hydroxyapatite column chromatography. One of the
peak substances exhibited thyrotropic activity about 10 times as
potent as bovine TSH. This fraction was reduced and subjected to
SDS-polyacrylamide gel electrophoresis. As a result, two bands were
observed at the positions of 14 and 17 kilodalton. When the reduced
TSH preparation was pyridylethylated and applied to reverse phase
HPLC, three main peaks were obtained. Amino acid sequence of N-
terminal 13 residues of the two peaks coincided with that of a-subunit
of bullfrog LH/FSH suggesting the difference in the sugar chains
between the two. Amino acid sequence of N-terminal 13 residues of
the remaining peak showed 46% homology with those of bovine and
porcine TSHBs. Thus, it was revealed that bullfrog TSH consists of B-
subunit with a considerable homology with mammalian TSHB and a-
subunit presumably similar to that of bullfrog LH/FSH.
ABSENCE OF METHYLATED TRH IN THE BULLFROG
(RANA CATESBEIANA) BRAIN.
K.Nakajima*, T.Yanagisawa*, S.Tanaka® and
S.Kikuyama*. *Dept. of Biol., Sch. of
Educ., Waseda Univ., Tokyo, 7Dept. of
Agricultural Chem., Utsunomiya Univ., Ut-
sunomiya, and *Inst. of Endocrinol., Gunma
Univ., Maebashi.
Recent finding that [1-Me-His*]TRH is
present in the carp brain prompted us to
ascertain whether the methylated TRHs exist
in the frog brain. Samples were prepared
from whole brain tissues of adult frogs,
climax tadpoles and prometamorphic tadpoles
by acid extraction. Each sample was sub-
jected to reverse-phase HPLC. Standard
samples of synthetic TRH, [1-Me-His~]TRH
and [3-Me-His*]TRH were eluted by MeCN with
concentrations of 4.5%, 4.8% and 6.9%
respectively. The eluates were assayed
using two antibodies against TRH, one show-
ing 100% crossreactivity to both methylated
TRHs and the other showing 3% and 43%
erossreactivities to [1-Me-His*]TRH and [3-
Me-His*]TRH. Both the distribution and
amount of TRH in the eluates of each sample
did not vary between the assays using the
two different antibodies. In both cases a
single peak with a similar immunoreactivity
appeared at the position similar to that
for synthetic TRH. Thus, it was concluded
that the brains of adult and larval
bullfrog do not contain any detectable
methylated TRHs.
EFFECT OF PARTIAL ABLATION OF
HYPOTHALAMIC PRIMORDIUM ON THE
ESTABLISHMENT OF BACKGROUND ADAPTATION.
Z. Kato, K. Kawamura and S. Kikuyama,
Dept. Biol., Schl. Educ., Waseda Univ.,
1-6-1 Nishi-Waseda, Shinjuku-ku, Tokyo
169-50, Japan. d
Our previous study indicated that in the
toad, Bufo japonicus, preoptic recess
organ (PRO) is the most probable
candidate for the center involved in the
background response. In order to confirm
this, experiment was conducted using the
embryos. Anterior part of the neural
plate, closely situated to the anterior
neural ridge, and prechiasmatic area were
manually removed at the open neurula and
the tail-bud stages, respectively. After
metamorphosis, they were tested for the
responsivenss to the background color.
The animals which did not respond to the
background but were permanently black,
tyrosine hydroxylase (TH)-positive
neurons in PRO did not develop or
developed poorly, whereas in
paraventricular organ and nucleus
infundibularis dorsalis, TH-positive
neurons developed normally. On the other
hand, in those showed a normal background
response, well-developed TH neurons were
observed in PRO as in the sham-operated
animals. These results demonstrate that
dopaminergic neurons in PRO is
responsible for the control of MSH
secretion in the toad.
1264 Endocrinology
IN VITRO RELEASE OF THYROXINE AND
THYROGLOBULIN FROM TADPOLE THYROID GLAND
WITH SPECIAL REFERENCE TO CYCLIC AMP
RELEASE
Shintaro Suzuki and Keiko Fujikura
Inst.of Endocrinol., Gunma Univ., Maebashi
To elucidate how cyclic AMP (cAMP)
(second messenger) is related to the
release of thyroid hormones and thyro-
globulin (TG) in metamorphosing tadpoles
we examined the release of cAMP from _ the
thyroid gland. Thyroid half lobes of Rana
catesbeiana tadpoles were incubated in
vitro in the medium (Amphibian Ringer
solution, 0.2% glucose, 0.1% BSA, 10-4M
theophylline) containing bovine thyroid
stimulating hormone (bTSH) or’ forskolin
(adenylate cyclase activator). Thyroxine
(Ts), TG and cAMP released into the
incubation medium were measured by radio-
immunoassay. Both bTSH and forskolin
stimulated the release ol Ts, but did not
stimulate the release of TG so much.
A considerable amount of TG was released
even from untreated thyroid gland. The
amount of T.4)released into the medium
increased depending on the concentration
of bTSH or forskolin. The release of cAMP
from the thyroid gland was also observed
when bTSH or forskolin stimulated the
release of T,4. These results suggest that
in the release of thyroid hormones
adenulate cyclase is an important trans-
ducer and TG-release is regulated by
another mechanism in the thyroid gland.
ARTIFICIAL INDUCTION OF NEOTENY IN
HYNOBIUS RETARDATUS DUNN II. FEMALE
SALAMANDER CAN ALSO SEXUALLY MATURE IN THE
LARVAL FORM.
M. Wakahara, Zool. Inst., Fac. Scei.,
Hokkaido Univ., Sapporo.
Hynobius retardatus Dunn, a hynobiid
salamander inhabited in Hokkaido, is a
unique species which had been reported to
show neoteny. Unfortunately, however, the
neotenic population is believed to be
extinct. We are thus trying to revive the
neoteny in the laboratory conditions.
A large number of newly hatched
laryae of Hynobius were reared in
different doses of thiourea (TU). All
larvae reared in TU-free and in lower
concentrations of TU metamorphosed
normally within 40-90 days of rearing.
Several larvae in medium concentrations
(0.01-0.04%) survived as long as 800 days
or over in an aquatic form. Histological
observations revealed a lot of bundles of
morphologically mature spermatozoa in the
testes of one year-old males, and full
grown oocytes filled with large yolk
platelets in the ovaries of two years-old
females, both of which had well developed
external gills and central fins. It is
concluded that even female individuals
become sexually mature without undergoing
metamorphosis in the laboratory
conditions. Therefore, it seems possible
to revive the neoteny in Hynobius.
CHANGES IN MONODEIODINASE ACTIVITIES DURING
FLOUNDER METAMORPHOSIS
E. G. de Jesus, M. Tagawa, and T. Hirano
Ocean Research Institute, University of
Tokyo, Nakano, Tokyo, 164
In mammals, thyroxine (Ty) is
considered as prohormone that is converted
to triiodothyronine (Tg) in order to exert
its biological effects. Ty can also
undergo monodeiodination to yield inactive
reverse ‘T (rT3). During flounder
metamorphosis, tissue Ty levels show a
dramatic increase while T3 levels increase
only slightly. However, T3 is several
times more potent than T4 in promoting
metamorphosis. Assays for the activity of
both 5- and 5’-monodeiodinases were
developed. The rates of both 5- and 5’-
monodeiodination were low during premeta-
morphosis, increased dramatically during
prometamorphosis, remained high through
metamorphic climax, and declined in post-
climax and juvenile fish. , These results
suggest increased turnover of both T, and
T3 during metamorphosis. oat was
previously reported to be absent from
teleost plasma; however, not only is rT
present in whole body homogenates o
flounder at all stages of metamorphosis,
but also changes in its concentration
closely paralleled those of Ty, and T3- The
importance of the monodeiodinase systems
and the regulation of thyroid hormone
levels during flounder metamorphosis will
be discussed.
EFFECTS OF THYROIDECTOMY AND REPLACEMENT
THERAPY WITH THYROID HORMONE ON THE FIRST
SKIN-SHEDDING IN HATCHLING SNAKES.
M. Yoshihara! T. Murakami’ and C. Oguro?
Dept. of Oral Physiol., The Nippon Dental
Univ., Niigata, 2Toyama Univ., Toyama.
It is well known that thyroid hormone
stimulates molting in the lizards. On the
contrary, it has been reported that
thyroid hormone inhibits skin-shedding of
adult snakes. In the hatchlings of the
snake, Elaphe quadrivirgata, the first
shedding takes place about one week after
hatching. In the present study, role of
the thyroid gland and thyroid hormone on
the first shedding of the hatchling snakes
was searched. Thyroidectomized hatchlings
did not shed the skin until 15 days after
hatching. In the sham-operated hatchlings,
the first skin-shedding takes place nor-
mally between 6 and 8 days after hatching.
Thyroidectomized hatchlings injected with
thyroid hormone also shed normally. On the
other hand, administration of control
saline in thyroidectomized hatchlings did
not cause the first shedding. These
results indicate that the thyroid hormone
is necessary for the first skin-shedding
in the hatchling snakes, contrasting to
the situation in the adults.
Endocrinology 1265
THE ANNUAL CHANGES IN SERUM THYROID HOR-
MONE CONCENTRATION OF BOBWHITES (COLINUS
VIRGINIANUS) REARED IN OUTDOOR CAGES.
M.Sato H.Sakai, S.Wakabayashi, M.Shioya*
and S.Sato*. Dept. of Biology, Nihon
Univ. School of Dentistry, Chiyoda-ku,
Tokyo 101 and *College of Agriculture and
Vet. Medicine, Nihon Univ., Fujisawa 252.
The relationship between the annual
shift in serum thyroid hormones and the
breeding and molting was examined.
Ten male and female pairs of bobwhites
hatched in March 1991 were housed in 10
cages under a natural daylight length and
blood samples were examined, from July
1991, once every few weeks, until
September 1992, together with egg laying,
molting and food intake.
The egg laying terminated in the middle
of August 1991 and was again observed
between May and August 1992. Molting of
the whole body began in August 1991 and
ended in December; in April, molting was
partially observed and quite obvious in
September 1992. No significant seasonal
change in serum T4 was observed in both
sexes. Serum T3, however, showed a
remarkable increase in both sexes starting
in August 1991 and the high values were
Maintained by the middle of April 1992,
lowered towards August of the same year.
These results indicate that molting begins
soon after the egg laying period, with
which the marked increase in T3 well
synchronized. This concurrence was
observed earlier in males than in females.
EFFECTS OF THYROIDECTOMY ON SEASONAL
BREEDING OF JAPANESE MONKEYS
M.Nozaki, K.Shimizu and F.Mitsunaga.
Primate Research Institute, Kyoto
University, Inuyama.
To examine the role of thyroid hor-
mones on the seasonal breeding of Japan-
ese monkeys (Macaca fuscata), sexually
mature females were thyroidectomized
(THX; n=6) in early December, during the
mid-breeding season, or they remained
thyroid intact (n=4). They were housed
indoors individually, and blood samples
were collected three times a week to
monitor gonadotropin and gonadal hormone
secretions. All animals ovulated during
the breeding season before operation.
THX resulted in significantly earlier
termination of the breeding season:
Ovulatory cycles of all THX monkeys were
terminated soon after THX. THX monkeys
exhibited significantly high levels of
plasma prolactin in comparison with
those of controls. Such high levels of
plasma prolactin in THX monkeys may be
attributed to the increased secretion of
hypothalamic TRH, which stimulates both
thyrotropin and prolactin secretion.
The present results suggest that thyroid
hormones are involved in the regulation
of the seasonal breeding of Japanese
monkeys directly or indirectly by
mediating the modification of prolactin
secretion.
ESTROGEN RECEPTOR-LIKE IMMUNOREACTIVITY
WAS DETECTED TRANSIENTLY IN THE FACIAL
NUCLEUS OF THE NEONATAL RAT.
S.Hayashi ! and M.Yokosuka?. _—! Dept. Anat. & Embryol.,
Tokyo Metropolitan Inst. Neurosci., Fuchu, Tokyo and 2 Dept.
Animal Reprod., Tokyo Univ. Agricul. & Technol., Fuchu,
Tokyo.
Estrogen receptor-like immunoreactivity (ER-LI) was
detected in the brain of newborn rats by immunohistochemistry.
It was detected not only in the hypothalamus, amygdala and
midbrain central gray, but also in the medio-basal subnucleus of
the facial nucleus. The antiserum (AS) used was raised in a
rabbit in our laboratory, as the antigen a fusion protein of rat
estrogen receptor (ER)-f-galactosidase construct produced in
Esherichia coli cells being used. Specificity of this AS has been
confirmed (Okamura et al., J.Endocr.,1992). The ER-LI in the
facial nucleus was detectable only in the newborn rat, while that
in the other part of the brain was detected also in the adult.
Thus, the expression in this nucleus is only transient. By
microinjections of a fluorescent tracer, fast blue, into the facial
muscles of the newborn rat, motoneurons in the subnucleus,
where the ER-LI was detected, were revealed to project their
terminals to the posterior auricular muscles. However, the
majority of motoneurons marked with the tracer did not contain
ER-LI. No clear sex difference in the ER-LI was detected in
this nucleus. Physiological roles of ER-LI in the facial nucleus
of the newbom rat are presently unknown. (Supported in part
by the Grants-in-Aids for Scientific Research to SH, No.
03640636, from the Ministry of Education, Science and Culture
of Japan).
ESTROGEN RECEPTOR (ER) EXPRESSION IN
MOUSE UTERINE EPITHELIAL CELLS IN VIVO
AND IN VITRO.
T. Sato, T. Iguchi, *Y. Ohta and N. Takasugi.
Grad. Sch. Integrated Sci., Yokohama City Univ.,
Yokohama; *Tottori Univ., Tottori.
Nuclei of stromal cells in the mouse uterus
showed a strong ER immunostaining on the day of
birth (0 day of age). In contrast, ER in uterine
epithelial cells appeared by day 5. In the uterine
epithelial cells, ER was induced 24 h after a
single injection of diethylstilbestrol (DES), 178-
estradiol (Eg), testosterone or tamoxifen (Tx)
given at O day. DES, Eg and Tx induced ER in
uterine epithelial cells in vivo in a dose-
dependent manner. A single injection of 3 ug
DES significantly stimulated cell division of the
epithelial cells in the uterus. In order to study
the role of stromal cells in ER induction by
estrogen, uteri of mice at 0 day were excised,
trypsinized and separated into epithelium and
stroma. The epithelial cells cultured on collagen
membrane in serum-free medium in the presence
or absence of Eg showed no ER immunostaining,
but the stromal cells showed ER immunoreaction
after 3 days in culture. When epithelial cells and
stromal cells were co-cultured on either side of a
collagen membrane, 15% of epithelial cells
showed ER immunoreaction. These results
indicate that 1) the ER induced by neonatal
injections of DES is functional; 2) ER expression
in the epithelial cells appears as a result of
interaction with stromal cells.
1266 Endocrinology
MITOTIC CHANGES IN THE UTERUS OF PREGNANT
RATS DURING THE DEVELOPMENT OF THE METRIAL
GLAND. , 2 2 1
Y.Ohta , T. Satoh” and T.Iguchi’. Dept.
of Biol., Tottori Univ., Tottori, Dept. of
Biol., Yokohama City Univ., Yokohama.
gland develops in the mesometrial triangle
of the uterus during the mid-pregnancy,
persisting after parturition. The distri-
bution of granulated metrial gland (GMG)
cells and. their mitotic activities were
investigated in the rat uterus during the
mid-pregnancy. In the mesometrial decidua,
GMG cells slightly increased in number
during Days 8-12 of pregnancy and then
decreased, while the cells appearing in the
mesometrial triangle on Day 9 continued to
increase untill Day 16, forming the metrial
gland. By contrast, the mitotic change in
the triangle was similar to that in the
decidua. The mitotic activities increased
drastically on Day 9 and then gradually
decreased toward Day 16. The activities
were very low within the gland. Immuno-
histochemical studies revealed that proge-
sterone receptor was located in decidual
and muscle cells and fibroblasts, but not
in GMG cells throughout the development of
the gland. These results suggest that GMG
cells proliferate under the control of
decidual cells and/or fibroblasts in both
the decidua and triangle and those pro-
liferated in the decidua migrate into the
triangle.
DAILY CHANGE OF SEX STEROID HORMONES IN
THE FECES OF HOUSE SWIFT (APUS AFINIS)
H.Sakai, M.Sato, S.Wakabayashi and
S.Ishii* Dept. of Biology, Nihon Univ.
School of Dentistry, Chiyoda-ku, Tokyo 101
and *Dept. of Biology, School of
Education, Waseda Univ., Nishi-Waseda,
Tokyo 169-50.
Out of the colony in Shizuoka City, 350
feces were gathered in 26 hours during the
feeding period, and the contained steroid
hormones were quantitatively examined.
The number of feces per hour gathered at
night was half that of day; the ratio of
feces in which hormones were detected was
70%, amounting to three-fold of that in
the day. Testosterone was detected in 42
samples, the hormone amount ranging from
7.6 to 118 pg; estradiol-17g was detected
in 65 samples, ranging from 0.04 to 13 pg.
Furthermore, progesterone was detected in
83 specimens, ranging from 29 to 221 pg.
In 8.6% of the specimens, estradiol and
progesterone were both detected. In 1.7%,
testosterone and progesterone were both
detected. I SHA testosterone and
estradiol. The former 2 cases of hormone
detection were observed both in the day
and at night; the last case was observed
only at night.
These results indicate that the amount
of hormones contained in feces do not vary
by the order of hours but significantly
different between day and night.
INHIBITORY ACTION OF MATERNAL TESTOSTERONE ON
THE REGULATION OF FETAL GROWTH
K. Tsutsui
Dept. of Rad. Biophys., Kobe Univ. School of Med., Kobe
Fetal growth depends on maternal testosterone in the
rat (Tsutsui, 1991), The present study was conducted
to examine the mechanism of testosterone action and
its physiological significance. Maternal plasma
testosterone concentrations increased from 14 days of
gestation, reaching a peak on 18 days, and decreased
during 18-21 days when active fetal growth took place.
Scattergrams showed a negative correlation between
circulating testosterone levels of pregnant mothers on
gestation day 21 and body weights of their fetuses.
Artificial maternal exposure to testosterone (10 or 13
mg/one silastic plate) during 14-21 days of gestation
significantly induced decreases not only in fetal
weight but also in placental weight on gestation day
21. Intrauterine administration of placental lactogen to
testosterone-treated pregnant rats for 7 days
restrained the testosterone effect. Not only
testosterone but also estradiol were effective for the
decreases in fetal and placental weights under the
same dose treatment, while no significant correlation
existed between endogenous estradiol of normal
mothers and the weight of their fetuses.
Dihydrotestosterone, a nonaromatizable androgen, was
inactive. These results suggest that 1) rapid fetal
growth during the second half of late pregnancy is
related to the decrease in circulating maternal
testosterone, and 2) maternal testosterone through its
conversion to estradiol decreases the placental
function and consequently inhibits fetal growth.
EFFECTS OF PLASMA ANDROGENS AND THEIR S-
METABOLITES ON THE MOUSE SUBMANDIBULAR
GLAND.
K.Sawada and T.Noumura
Dept. Regulat. Biol., Fac. Sci., Saitama
Univ. Urawa
In the mouse submandibular gland, sex
difference is evident on 30 day, when the
granular convoluted tubules (GCT) in the
male gland rapidly grow in response to
dramatically increased levels of circu-
lating testosterone (T) and 50-dihydrotes-
tosterone (DHT). We studied in vitro
metabolism of these androgens by the
gland and the effects of their metabolites
on the gland. The glands of both sexes did
not convert T to any metabolites, but DHT
to 5a-androstane-3Q4,1768-diol (3a-diol;
8%) and SG@-androstane-3@,176-diol (38 -
diol; 1%). Neonatally-castrated mice re-
ceived daily treatment with these four
androgens for 1-10 days from day 20. The
relative occupied area (ROA) of GCT
increased in both sexes by treatment with
3d-diol or OHT for 4 days, but by 38-diol
or T for 10 days. The ROA gain after 10
days was in the order of DHT = 3@-diol >
T > 38-diol. The mitotic activity of GCT
increased in both sexes by 3Q-diol or DHT
for 4 days, but in only males by 38@-diol
or T for 10 days. The results suggest that
these DHT metabolites have androgenic
effects on the gland growth and particu-
larly 3Q@-diol has a potency similar to
DHT.
Endocrinology 1267
ANDROGEN REGULATES 8-ACTIN mRNA EXPRESSION
IN THE MOTONEURONS OF LUMBAR SPINAL CORDS
IN ADULT MALE RATS.
A. Matsumoto (1), Y. Arai (1), A. Urano
(2) and S. Hyodo (3). (1) Dept. Anat.,
Juntendo Univ. Sch. Med., (2) Ocean Res.
Inst., Univ. Tokyo, (3) Fac. Art & Sci.,
Univ. Tokyo, Tokyo.
Androgen has been reported to play a
key role in reorganizing the neuronal
elements and synaptic connections in
androgen-sensitive motoneurons in the
spinal nucleus of the bulbocavernosus
(SNB). As one step to clarify molecular
mechanisms of androgen for neuronal plas-
ticity, we examined androgenic regulation
of the expression of ®-actin mRNA in the
SNB motoneurons of adult male rats. Adult
male rats (Wistar) were castrated and
implanted with silastic tubes containing
testosterone (T) or nothing. Animals were
sacrificed 4 weeks later. A complementary
DNA encoding chick B-actin was applied to
in situ hybridization on paraffin sections
of the lumbar spinal cords. Autoradio-
Sraphic signals for B-actin mRNA were
found to be localized on the somata and
proximal dendrites of SNB neurons. The
number of signals per neuron in castrates
was significantly smaller than that in
controls. The value in castrates given T
was not significantly different from that
in controls. These evidence suggests that
androgen regulates expression of B-actin
gene in the SNB motoneurons.
SEASONAL CHANGES OF BODY WEIGHT, GONADS
AND HORMONES IN DJUNGARIAN HAMSTERS KEPT
OUTDOORS.
A. Masuda and T. Oishi. Dep. of Biology,
Fac., of Sci., Nara Women's Univ., Nara.
The Djungarian hamster is an animal
which shows a distinct annual cycle in
some physiological parameters.
In order to investigate the seasonal
changes in detail under natural
conditions, we kept male and female
hamsters at the veranda of our university
building. In most hamsters, body weight
decreased in winter and started to
increase in spring. Food intake per body
weight was high in winter and water intake
per body weight was high in summer and
winter. The brown and white adipose
tissues did not show clear differences
among seasonal groups. Muscle weight in
the winter group was less compared with
that in the summer group. The weights of
testes and accessory glands were small and
plasma testosterone level was low in the
winter group. Thus the seasonal changes
of testes weight and muscle weight showed
a similar tendency. The weight of heart,
kidney and liver per body weight and the
length of intestines were larger in the
winter group than in the summer group.
This is in accordance with the changes in
plasma T3 level and food intake,
suggesting the involvement of metabolic
activity.
CHANGES IN PROTEIN EXPRESSION IN
PREGNANCY-DEPENDENT MAMMARY TUMORS
(PDMT) DURING GROWTH AND REGRESSION IN
GR/A MICE.
Y. Goto, T. Iguchi, H. Nagasawa’ and N. Takasugi.
Grad. Sch. Integ. Sci., Yokohama City Univ.,
Yokohama; *Exp. Animal Res. Lab., Meiji Univ.,
Kawasaki.
The GR/A mouse is characterized by the
development of pregnancy-dependent mammary
tumors (PDMT). PDMT appear after the middle
of pregnancy, reach maximal size at the end of
pregnancy and regress soon after parturition.
Mitotic index in the epithelial and stromal cells of
PDMT decreased before parturition. Using 2-
dimensional electrophoresis, protein expression
in PDMT was studied during growth and
regression. On Day 20 of pregnancy, 9
polypeptide spots increased in intensity, and 5
protein spots appeared newly in PDMT. Three
protein spots increased in these intensity, and 2
new spots appeared at parturition. Four of these
19 proteins were not observed in normal
mammary glands. The growth rate of PDMT
transplanted into progesterone-treated mice was
higher than in mice treated with estradiol alone
or with a combination of estradiol and
progesterone. The expression of specific
proteins was observed in PDMT transplanted into
host mice receiving estradiol or progesterone.
These findings suggest that expression of specific
proteins may be related to growth and regression
of PDMT.
EFFECT OF OVARIECTOMY ON PROTEIN
EXPRESSION IN REPRODUCTIVE TRACTS OF
MICE.
A. Suzuki, Y. Abe, T. Iguchi, Y. Ohta* and
N. Takasugi. Dept. Biol., Yokohama City Univ.,
Yokohama; *Dept. Biol., Fac. General. Education,
Tottori Univ., Tottori.
Proliferation and regression of the vagina and
uterus in rodents are controlled by ovarian steroid
hormones, especially estrogen. Mouse vagina and
uterus regress after ovariectomy. In vagina and
uterus examined 1, 2, 3, 5, 7 and 20 days after
ovariectomy, their weights, epithelial thickness and
the number of epithelial cell layers were
significantly reduced when compared with those in
intact mice at estrous stage. No significant
differences in these parameters were found between
estradiol-implanted ovariectomized mice and intact
estrous mice. Protein expression in vagina and
uterus of ovariectomized mice was examined by
two-dimensional polyacrylamide gel electrophoresis
in the regressing vagina and uterus. Seven and 6
proteins in vagina and uterus, respectively, showed
the highest expression 2-3 days after ovariectomy.
Zymography revealed that expression of
metalloproteinases increased 2-3 days after
ovariectomy in vagina and uterus. Matrin, one of
the metalloproteinases, was secreted asa
proenzyme in the uterus and as an active form in
the vagina. These results suggest that
metalloproteinases and some other proteins
participate in regression of the vagina and uterus in
mice after ovariectomy.
1268 Endocrinology
TGF-8 AND INHIBIN EXPRESSION IN THE
DEVELOPING RAT GONADS
S.Koike! and T.Noumura. Dept. of Regul. Biol., Fac. of
Sci., Saitama Univ., Urawa. ‘Pathol. & Toxicol. Res.,
Upjohn Pharmaceuticals Ltd., Tsukuba.
To determine the expression of TGF-f and inhibin
in the developing Sprague-Dawley rat gonad, gonads
from the gestational day (GD) 13 through the postnatal
day (PD) 21 were fixed in Metakarn solution and
immunohistochemically stained with two polyclonal
antibodies, one against porcine platelet TGF-f and the
other against bovine follicular fluid inhibin-a subunit,
respectively.
TGF-f was detected in male and female germ
cells throughout the perinatal period. Moderate or
marked staining was observed in male germ cells from
GD 16 to PD 5 and Leydig cells from GD 16 to PD 11,
while in female germ cells from GD 21 to PD 11 and the
interstitial cells since PD 11. In both sexes, the
mesonephric tubules faintly stained from GD 13 to 18.
On the other hand, inhibin-a subunit was weakly
expressed in Sertoli/supporting cells on GD 14 and 15,
and markedly in Leydig cells from GD 17 through 20
and granulosa cells on PD 21.
These results indicate that the expression of
TGF-8 and inhibin-a is stage-specific during gonadal
development and that both growth factors may
participate in gonadal and extragonadal differentiation.
EFFECT OF NEONATAL EXPOSURE TO
DIETHYLSTILBESTROL ON PELVIS AND FEMUR IN
MALE AND FEMALE MICE.
S. Nobata, T. Sato, T. Iguchi and N. Takasugi.
Dept. Biol., Yokohama City Univ., Yokohama.
Neonatal treatment of male and female mice
with diethylstilbestrol (DES) causes irreversible
changes in reproductive organs. Migliaccio et al.
(1992) reported that total calcium in lumbar
vertebrae and in femur is reduced in neonatally
DES-exposed female mice. We studied changes in
the pelvis and the femur of male and female mice
given 5 daily injections of 3 ug DES from the day of
birth. Both pelvis and femur of 12-, 15- and 18-
month-old neonatally DES-treated mice were lighter
than in controls. Total amount of calcium and
phosphorus in both bones of neonatally DES-
treated 12-, 15- and 18-month-old mice were less
than in the age-matched controls. Total amount of
calcium and phosphorus in pelvis and femur of 12-
month-old, but not 2-month-old, female mice
exposed neonatally to DES were less than in
controls. These results indicate that neonatal DES
exposure causes irreversible changes in bones of
aged male and female mice.
3-DEHYDROECDYSONE-38-REDUCTASE IN THE
LARVAL HEMOLYMPH OF THE SILKWORM, BOMBYX
MORI. T. Momura and S. Sakurai, Dept.
Biology, Kanazawa Univ., Kanazawa, Japan.
3-Dehydroecdysone (3DE) is the major
ecdysteroid secreted from insect prothora-
cic glands and exhibits no biological
activity. Since 3DE is converted to
ecdysone by 3f-reductase found in
hemolymph and require NADPH as co-factor,
the change in the enzyme activity may
directly affect on the change in the
concentration of ecdysteroids with
biological activity, ecdysone and 20-
hydroxyecdysone. Therefore, we first tried
to purify the enzyme so as to understand
the control mechanism of the enzyme
activity. The enzyme was purified after 5
steps of purification, but the purified
sample gave two bands on SDS-PAGE.
Analysis with amino acid sequencer gave
only one amino acid sequence, indicated
that these two bands on SDS-PAGE was
originated from a single protein or N-
terminal of either of the bands is
blocked. Enzymatic study showed that the
optimum pH is 6.6, similar to the pH of
larval hemolymph, and the enzyme requires
NADH (Km, 5. 4yM) or NADPH (Km, 0.9yM) .
PRECISE DETERMINATION OF HEMOLYMPH
ECDYSTEROID TITER IN THE LAST LARVAL
INSTAR OF THE SILKWORM, BOMBYX MORI
S. Sakurai, M. Kaya and S. Satake,
Dept. Biology, Fac. Science, Kanazawa
Univ. Kanazawa, Japan.
Hemolymph ecdysteroid titer was
precisely determined by ecdysteroi radio-
immuno assay from the onset of the fifth
instar through pupation. The detectable
limit of ecdysteroid concentration was
approximately 50 pg/ml hemolymph. In the
fifth instar, some developmental events
can be observed, such as head critical
period found on day 4, spinneret
pigmentation on day 6 and gut purge on day
7. The ecdysteroid concentration exhibited
daily change and an increase in
ecdysteroid titer was observed prior to
each developmental event described above.
Such titer was lower than that examined so
far and 10-20 ng/ml was enough to induce
gut purge. Though daily release of PTTH
has not been demonstrated, the present
data strongly indicated that PTTH may be
secreted during a specific gate of every
day and such daily change in ecdysteroid
titer may integulate the preparation for
pupation in various tissues in larvae.
Endocrinology 1269
IMMUNOBLOT ANALYSES OF THE MATERIALS IMMU-
NOREACTIVE TO THE BOMBYXIN-II ANTIBODY
PRESENT IN THE HEMOLYMPH OF BOMBYX MORI
DURING PUPA-ADULT DEVELOPMENT
H.Saegusa!, A.Mizoguchi!, A.Suzuki2, and H. Ishizaki!
IDept. of Biol., School of Sci., Nagoya Univ., Nagoya;
2Dept. of Agr. Chem., Fac. of Agr., Univ. of Tokyo,
Tokyo
We previously obtained a monoclonal antibody against
natural bombyxin-lI (bombyxin-II antibody) and examined
changes in the titer of the immunoreactivity in the
hemolymph during Bombyx development by a radio-
immunoassay using this antibody. It was later found that
the bombyxin-II antibody recognized not only bombyxin
but also another 8-kD component of unknown nature. In
this study, materials immunoreactive to the bombyxin-II
antibody were immunoprecipitated using this antibody and
immunoblotted with the same antibody. Using this system,
we differentiated bombyxin and the 8-kD component in
the immunoreactive material at each stage. In females,
only the 8-kD component was detected throughout the
pupa-adult development. In males from pupation to 1 day
before eclosion, the 8-kKD component was the major
component, but in newly ecdysed adults, bombyxin was
predominant. Thus, bombyxin is thought to be involved in
the regulation of functions specific to the male adults.
The nature of the 8-kD component is being studied.
NEUROSECRETORY CELLS PRODUCING SAMIA
BOMBYXIN-RELATED PEPTIDES (SBRP)
IMMUNOHISTOCHEMICAL LOCALIZATION AND
DEVELOPMENTAL CHANGE.
Y.Yagi!, A.Mizoguchi!, K.Kojima2, K.Nagata~,
A.Suzuki2 and H.Ishizaki!. !Dept. of Biol., School of Sci.,
Nagoya Univ., Nagoya and 2Dept. of Agr.Chem., Fac. of
Agr., Univ. of Tokyo, Tokyo.
Bombyxin is an insulin-like peptide produced by the
Bombyx brain which shows prothoracicotropic activity
when administered to debrained pupae of Samia cynthia
ricini. Samia genes which code for bombyxin-related
peptides have been cloned and characterized.
We chemically synthesized one of these peptides
(SBRP-A1) based on the amino acid sequence deduced
from the gene structure, and raised a mouse antiserum
against the synthetic SBRP-Al. We performed an
immunohistochemical study using this antiserum on
Samia from the (first-instar larva to adult.
Immunoreactivity was observed in the medial
neurosecretory cells of brain, their axons, and axon
terminals in the periphery of the corpora allata. The
number of the immunoreactive cells were counted. The
maximal number of the immunoreactive cells was 16 per
brain hemisphere. The stainability and the average and
maximal number of the immunoreactive cells did not
change appreciably during the development.
RELATIONSHIP BETWEEN BOMBYXIN-IMMUNOREACT-
IVE PEPTIDE AND SMALL PROTHORACICOTROPIC
OSIVO SOUP LE) IN MANDUCA SEXTA.
A.Mizoguchi’ and L.I.Gilbert?. IDep. of
Biology, Fac. of Sci., Nagoya Univ., Nagoya
and 2Dep. of Biology, The Univ. of North
Carolina, Chapel Hill, NC, USA.
The Manduca small PTTH(Mr,7kDa) is
similar in size and biologic activity to
bombyxin(Mr,5kDa), a brain neurohormone of
Bombyx mori which manifests PTTH activity
on the prothoracic glands(PG) of Samia
cynthia ricini. The presence of a
bombyxin-like peptide in Manduca brain was
demonstrated by immunohistochemistry and
immunoblotting using a bombyxin antibody.
To investigate the relationship between the
small PTTH and bombyxin, a Manduca brain
extract was chromatographed and the
resulting fractions were examined for both
PTTH activity and bombyxin immunoreac-
tivity(BIR). The PTTH activity was
assessed by an in vitro PG assay, and the
BIR by immunoblotting. Among the fractions
obtained by gel filtration with Sephadex
G-50, the distributions of both activities
were overlapped with slight difference.
However, when the fractions containing the
BIR were subjected to an anion exchange
FPLC, the BIR bound to the column, while
the PTTH activity did not. The BIR was
eluted with an increased NaCl concen-
tration. These results indicated that the
Manduca neuropeptide that was recognized by
the bombyxin antibody was not identical to
the small PTTH.
SPECIES-SPECIFICITY IN THE ACTION OF
BIG AND SMALL PROTHORACICOTROPIC HORMONE
OF SEVERAL SPECIES OF THE SWALLOWTAILS.
I.Yokoyama and K.Endo. Environ. Biol.
Lab., Biol. Inst., Fac. of Sci., Yama-
guchi Univ. Yamaguchi.
Lepidopteran insects have two mole-
cular forms of (big and small) prothora-
cicotropic hormone (PTTH) activating the
prothoracic glands (PGs) to secrete ecdy-
steroid(s), which is essential for growth
and metamorphosis in insects.
Extracts containing the big and
small PTTHS were made from pupal brains
of several species of papilionids, Papilio
xuthus, P. machaon, P. bianor and P.
helenus. The PTTHS were separated by
centrifuging the extracts with a ultra-
filter, UFC3LGC, passing molecules smal-
ler in the sizes than M.W. 10,000.
The PGs from 2-day-old 5th instar
larvae of the same or other species in-
sects were incubated for 2 hr in Grace's
medium containing the big or small PTTH
extract. The titir of ecdysteroids
secreted in the incubation medium were
assayed by RIA. Activity of the big and
small PTTHs on the PGs were quantified by
activation ratio (Ar) of 5-6 pairs.
The big and small PTTHs of papilio-
nids examined activated the PGs of
the same and other species of 5th-instar
papilionid larvae in vitro.
1270 Endocrinology
ENTRAINMENT OF THE CIRCADIAN CLOCK GATING
PROTHORACICOTROPIC HORMONE SECRETION IN
THE ASIAN COMMA BUTTERFLY, POLYGONIA C--
AUREUM L.
K.Endo, N.Kusumuto, Y.Ito and N.Matsu-
shima. Biol. Inst., Fac. of Sci., Yama-
guchi Univ. Yamaguchi.
Prothoracicotropic hormone secretion
inducing larval ecdysis from the 4th to
5th instar preceds the larval ecdysis by
32 hr at 25°C. The PTTH secretion was
gated by a circadian clock in Polygonia.
The acrophase-time of PTTH secretion
which was obtained by subtracting 32 hr
from the time of larval ecdysis came to
on an almost parallel line with a line
connecting the mid points of 2-hr to 20-
hr light periods in 24-hr LD cycles.
Groups of insects reared under 2-hr
to 18-hr light photoperiodic regimens at
25°C were transferred to continuous light
or to contimuous dark condition at 25°C
and the acrophase-time of PTTH secretion
was obtained in these groups addition to
the other larval groups raised from the
egg stage under resonant conditions con-
sisting of an 8-hr light and an various
length (2-hr to 64-hr) dark periods.
The results indicated that the circa-
dian clock gaing PTTH secretion may be set
twice a day, at dawn and dusk. At dawn the
circadian clock may be reset at CT 0:00,
and again set at dusk to give the same
time (CT 18:00) amid the dark period of
24-hr LD cycles.
HOW DOES PARASITIC WASP INDUCE GROWTH-
BLOCKING PEPTIDE IN THE PARASITIZED
ARMYWORM LARVAE?
Y. Hayakawa, Biochem. Lab., Inst. of Low
Temp. Sci., Hokkaido Univ., Sapporo.
Last instar larvae of the armyworm
parasitized with the parasitoid wasps,
Cotesia kariyai, do not initiate
metamorphosis and, ultimately, the wasp
larvae emerge from the host larvae about
10 days after parasitization. The
developmental arrest can be reproduced by
injection of parasitoid ovarian calyx
fluid containing the symbiotic virus
(polydnavirus) which is normally injected
by female wasps into the host at
Oviposition. A peptidergic factor,
g€rowth-blocking peptide (GBP), has been
purified from the larval hemolymph of the
parasitized armyworm. Injection of GBP
into unparasitized last instar larvae of
the armyworm clearly retards larval growth
and, consequently, delays the onset of
pupation of the larvae. Recently, it has
been demonstrated that GBP exists in
plasma of the virus-injected unparasitized
last instar larvae and also in plasma of
the penultimate instar larvae of the
armyworm. Therefore, it is reasonable to
propose that infection of polydnavirus
activates GBP gene expression, and the
production of GBP retards a normal
development of last instar armyworm
larvae.
ECDYSTEROIDS DURING EARLY EMBRYOGENESIS IN THE
SILKWORM, BOMBYX MORI.
H. Sonobe’, T. Masumoto’, Y. Mamiya’, T. Kanda’ and
T. Tamura’. ‘Dept. of Biol., Konan Univ., Kobe.
*"Natl. Inst. Seric. Sci., Tsukuba.
Ovaries in the silkworm are the site of
biosynthesis and accumulation of ecdysteroids, and
these accumulated ecdysteroids are transported
into eggs.
First, ecdysteroid content of diapause eggs and
non-diapause eggs was analyzed by both RIA and
reverse phase HPLC. Nine free ecdysteroids and
their conjugated forms were detected throughout
early embryogenesis. In diapause eggs, most free
ecdysteroids remained at original levels, but
conjugated forms began to increase with the onset
of the diapause. In non-diapause eggs, most free
ecdysteroids, including 20-hydroxyecdysone, began
to increase as embryogenesis proceeded. In
contrast, conjugated forms remained at their
original levels.
Next, in order to examine the function of free
ecdysteroids in the eggs, 20-hydroxyecdysone was
injected into 20-hr prospective diapause eggs.
Their developmental fate was changed from the
diapause type to the non-diapause type. This fact
strongly suggests that the elevation of the titer
of 20-hydroxyecdysone is needed to advance
embryonic development of the silkworm.
PHYSIOLOGICAL SIGNIFICANCE OF
3-DEHYDROECDYSONE(3dhE) FROM CRAYFISH,
Procambarus Clarkii
M.Ikeda, Y.Naya. Suntory Institute for Bioorganic
Research (SUNBOR), Osaka.
Finding the dominant ecdysteroid 3dhE from P.
clarkii, uponin vitro culture of Y-organs(1), led us
to inquire what the physiological function of 3dhE is.
After bilateral eyestalk ablation, the production of
3dhE in Y-organs was increased prior to ecdysone(E)
and accompanied by an increase of 20-hydroxyecdys-
one(20E) in hemolymph.The hormonal response of
3dhE, upon injection into P.Clarkii, was comparable
to those of E and 20E. When a large amount of 3dhE*
was injected into crayfish, most of the unchanged ma-
terial was excreted within 1h and then its metabolite*
(3-epi-E,3-epi-20E,ecdysteroids'conjugates etc.) was
observed in the excretion. The major radio isotope in
the body was found 3h after administration in the
epidermal tissues(with carapace), and after 72h in the
hepatopancreas. The epidermal isotope was found to
be comprised of 3dhE,E,20E,3-epi-E,3-epi-20E,polar
ecdysteroids and conjugates efc.. Ecdysteroids* were
separated from the conjugates* by enzymatic digestion
(Helix pomatia ). Our results concluded that 3dhE is
the intimate precursor of E and results in the produc-
tion of 20E in P. Clarkii.
1) H.Sonobc, M.Kamba, K.Ohta, M.Ikeda and Y.Naya,
Experientia, 47, 948(1991).
Endocrinology 1271
EXPRESSION PATTERN OF ECDYSTEROID
RECEPTORS IN MUSCULAR SYSTEM DURING”
METAMORPHOSIS OF DROSOPHILA
MELANOGASTER
K-I. Kimura!, D.A. Currie? and J.W. Truman
1Lab. Biol. Hokkaido University of Education,
lwamizawa, 2Dept. Zoology, Univ. Washington, USA
In holometabolous insects such as Drosophila
melanogaster, adult musculature is rebuilt during
metamorphosis. Many larval muscles die at
beginning of metamorphosis. However, some larval
muscles persist during metamorphosis. These
persisting muscles regress once but then regrow to
transform into adult ones. Also, adult specific
muscles are newly formed from adult myoblasts.
These processes should be regulated by ecdysteroid
hormone. Ecdysteroid hormone binds to the receptor
(ecdysteroid receptor; EcR) and regulates the gene
expression. In Drosophila, the EcR gene encodes
three EcR isofrom, EcR-A, EcR-B1 and EcR-B2. We
examined the expression pattern of EcR-A and EcR-
B1 in muscular system during metamorphosis, using
isoform-specific antibodies. Both of EcR-A and -B1
were expressed in a Stage-specific manner during
metamorphosis, respectively, that would correspond
to the processes of muscle development. These
results suggest that different combinations of EcR
isoforms affect the developmental responce in
different processes of muscle developmet.
ROLE OF ECDYSTERONE IN ALTERATION OF NERVOUS
SYSTEM DURING METAMORPHOSIS IN BOMBYX MORI.
H. Tsujimura. Lab. Biol., Tokyo Univ. Agri.
Tech., Fuch, 183 Tokyo.
Hormonal requirement for the alteration of
the ventral nerve cord and motor neurons dur-
ing metamorphosis is examined by injecting to
the pupa the ecdysterone and an imidazole
compound, 1-benzy1-5-([(E)-2,6-dimethy1-1,5-
heptadienylJimidazole (KK-42), which inhibits the
ecdysone biosynthesis in the prothoracic gland.
Many changes in the ventral nerve cord and
motor neurons were inhibited by injection of
KK-42 just after the pupal ecdysis, suggesting
that they are controlled by a peak of ecdy-
sterone titer in the pupal hemolymph. Results
from several injection experiments show that
the connective shortening and ganglionic fusion
are stimulated by low titer of ecdysterone but
inhibited by high titer of ecdysterone. On the
other hand, low and high titer of ecdysterone
stimulate the programmed cell death of certain
larval motor neurons and the disappearance of
larval dendrites and regrowth of adult ones in
other motor neurons which survive and become
adult neurons.
These results show that alteration of ecdy-
sterone titer in the pupal hemolymph controls
the change of the CNS but there are 2 distinct
mechanisms to respond to the hormone, which
controls different part of the change.
REGULATION OF DIAPAUSE HORMONE SECRETION
IN THE SILKWORM, BOMBYX MORI:A TRY USING
LASER MICROBEAM TREATMENT.
I. Shimizu. Center for Ecological Research
Kyoto University, Kyoto.
The diapause hormone of the silkworm
is believed to be produced in and secreted
from neurosecreatory cells located in the
suboesophageal ganglion(SG). However, I
found, extirpataion of SG from the
silkworm pupae which had been destined to
produce non-diapause eggs resulted ina
production of diapause eggs:the treatment
caused a production of diapause in eggs
which developed at the later pupal stage.
On the other hand, extirpation of Brain
(CC-CA)-SG complex from the non-diapause
producer pupae did not produce diapause
eggs as sham controls. These observation
pose a question about the established
thinking mentioned above about the dia-—
pause hormone. Another new idea(produ-
ction in SG and secreation from CC-CA) is
proposed. Further a new try using laser
microbeam to invesigate the hormone regu-
lation mech- anism is reported pre-
liminary.
EXOCYTOTIC RELEASE OF NEUROPEPTIDES IN THE BRAIN-MIDGUT
ENDOCRINE SYSTEM OF INSECTS
Y. ENDO
Dept.of Appl.Biol., Kyoto Institute of Technology,
Matsugasaki, Sakyo, Kyoto
In order to clarify the significance of non-
synaptical release of neuropeptides, I have examined the
brain-midgut endocrine system of insects, Periplaneta
americana and Galleria mellonella, at the level of
morphology by immunohistochemistry, scanning and
transmission electron microscopy. The tissues were
isolated in the saline solution and the exocytosis was
stimulated by high Kt and Ca?* solution (120mM KCl,
6mM CaC], 30mM NaCl). After the fixation of tannic
acid-containing glutaraldehyde and Os0x, omega-shaped
figures were detected by electron microscopy. In the
retrocerebral neurohemal organs (corpora crdiaca and
allata), the exocytosis of neuropeptides occurred not
only on the free surface of neurites, but also on the
surface of neurites facing the target cells such as the
glandular cells. In the muscle layer of gut,
neuropeptides were also released non-synaptically.
These results were comparable to the phenomenon of
non-synaptical neurotransmitter release in the
peripheral autonomic nerves in mammals! 2%). Some of
them might be not a neurotransmission or neurosecretion
in nature, but the other including neurotrophic or
neuro-survival factors.
1)Y.ENDO et al. (1991) Neurosci. Lett., 126:60-62.
2)Y.ENDO (1988) Zool.Sci., 5: 965-971h
3)Y.ENDO (1988) Arch.Histol.Cytol., 51:489-494
1272 Endocrinology
DISTRIBUTION OF NEUROSECRETORY CELLS IN
SYNGANGLION OF THE ADULT FEMALE TICK,
HAEMAPHYSALIS LONGICORNIS, WITH REFERENCE
TO THE INCREASE OF STAINING INTENSITY IN
INACTIVATED TICKS UNDER LOW TEMPERATURE.
N. Okura, T. MOri and S. Shiraishi.
Zoological Lab., Fac. Agriculture, Kyushu
Univ., Fukuoka.
Paraldehyde thionin (PAT) staining (a
modified technique of Gomori’s paraldehyde
fuchsin) revealed positive neurosecretory
cell bodies (34 in number) throughout the
cortex of synganglion in the adult female
tick. These cell bodies were distributed
in sub-groups within 11 neurosecretory
centers similar to those of other ixodid
ticks reported so far.
By exposure to the low temperature
(10°C, for i10days), active ticks were
inactivated. There was a conspicuous dif-
ference in staining intensity of certain
cell bodies between active and inactivated
ticks. Although a few cell bodies of the
frontal neurosecretory center (one of the
11 centers) in active ticks were hardly
stained, those in inactivated ticks had
remarkably high intensity. Such high in-
tensity suggests that accumulation of PAT
positive neurosecretory materials within
the cell bodies is caused by cessation of
neurosecretory discharge. Thus, this neu-
rosecretory center may play some regulato-
ry roles concerning temperature dependent
activity.
PREMATURE EGG-RELEASING SUBSTANCE (PERS) IN
ESTUARINE CRABS
M. Saigusa, Coll. of Liberal Arts & Sci.,
Okayama Univ., Okayama
The eggs of most decapod crustaceans
are attached to ovigerous hairs by a funi-
culus, and are ventilated by the female.
When the embryonic development is complete,
hatching occurs. This study focuced on a
substance that remained in the medium
(hatch water) where a larval release had
occurred. When another ovigerous female
contacted with this hatch water, she dro-
pped within a few days all eggs without
hatching. This egg-releasing activity clea-
rly appeared associated with hatching, and
the effect was eliminated by boiling and
tripsin, which suggests that this substance
(PERS) is protain. Gel filtration of hatch
water revealed that the molecular weight
of PERS is about 20,000 in Sesarma haema-
tocheir, and about 29,000 in Sesarma
pictum.
This substance would function, by
nature, as the deposition of remnants
remained on the ovigerous hairs after the
larval release. But the primary function
must have an immediate connection with
larval hatching. It might be a hatching
enzyme in Crustacea,
OVARIAN FACTOR(S) INDUCIBLE SECONDARY
SEXUAL CHARACTERS IN ARMADILLIDIUM
vulgare (ISOPOD).
S. Suzuki* and K. Yamasaki+#«*
*Biol. Lab. Kanagawa Pref. Col., Yokohama,
**Dept. Biol. Tokyo Metropol. Univ.,Tokyo.
In male A. vulgare, the androgenic
gland hormone is responsible for sex
differentiation, but in female there is no
direct evidence of the existence of sex
hormone controlling development of sexual
characters. In order to elucidate the
mechanism of sex differentiation in female,
oostegite formation was selected as the
index for the expression of secondary
female character in our experiments.
Young animals of both sexes (5th stage
after the 4th molt) were ovariectomized or
andrectomized by removing all internal
reproductive organs. These gonadectomized
animals attained to adult size after six
months but did not form oostegites.
However, they could form oostegites by the
injection of the extract of vitellogenic
ovaries. The ovarian extract was dialyzed
against distilled water. The dialysable
fraction was active for the induction of
oostegite formation. The optimal amount
for the activity was 1/2 ovary equivalent.
These results suggest that a vitello-
genic ovary contains the factors which are
responsible for sex differentiation of the
female A. vulgare. The isolation and
characterization of the ovarian factors
are now in progress.
PURIFICATION AND SEQUENCE ANALYSIS OF
ANDROGENIC GLAND HORMONE OF THE TERRESTRI-
AL ISOPOD,._ARMADILLIDIUM VULGARE.
H.Nagasawal, ¥-Hasegawa 5 K.Haino® and
Y.Katakura*. locean Res. Inst., Tokyo
Univ. Tokyg, 2Dept. of Biol., Keio Univ.
Yokohama, “Dept. of Biol., Tokyo Metropol-
itan Univ. Tokyo, “Dept. of Bioengineer-
ing, Soka Univ. Tokyo.
In crustaceans, male sexual characters
are induced by a peptide hormone, andro-
genic gland hormone (AGH), which is pro-
duced by androgenic glands (AGs). AGH was
first purified from whole male reproduc-
tive organs of the terrestrial isopod,
Armadillidium vulgare, ang some chemical
properties were clarified yy Until recent-
ly, however, no sequence information has
been obtained.
In the present study, AGH was purified
again from 16,000 male reproductive or-
gans, and finally about 23 pe (1.3 nmol)
of AGH was obtained. Because the N-termi-
nus of AGH was blocked, it was digested
separately with lysyl endopeptidase,
chymotrypsin and endoproteinase Glu-C. The
resulting peptide fragments were se-
quenced. Though the complete amino acid
sequence of AGH has not yet been obtained
by aligning the sequences, the following
partial sequences were identified; MTTQMG-
KLR, LKVVGQDSNEIHFRVK, KSYSERVGVPVASLRFLF-
DGRRINDEETPK and EMENDDVIEVYQEQTG.
1) Y.Hasegawa et al., Gen. comp. Endocri-
nol, Gi, LOL L987).
Endocrinology 1273
CALC1 TON IN- IMMUNOREACTIVE SUBSTANCE
PRESENT IN THE CELLS OF MIDGUT CAECUM OF
AMERICAN CRAYF1!SH
Y. Nosé, N. Suzuki and Y. Sasayama
Dept. Gre Iul@uhs 5 Fac. Ge SOAs » Toyama
Umiv., Toyma 930, Japan
Calcitonin induces a hypocalcemia in
mammals. Recently, we found calcitonin-
immunoreactive cells in the midgut caecum
in some crustaceans. In the present study,
the positive substance contained in those
cells of the American crayfish
(Procambarus clarki) was studied. At
first, the midgut caeca were homogenized
with 0.1 M acetic acid. The homogenate was
separated centrifugally to two fractions
(molecular weights (MW): 3,000-10, 000 and
10, 000-30, 000). These fractions were
subjected to SDS-PAGE, to estimate the MW
of the substance. For this purpose,
immunoblotting method using anti-salmon
calcitonin antiserum was applied. As a
result, two positive bands were found
around 4,000 MW and 17,000 MW. In the next
step, fractions containing these extracts
were injected into the pericardial cavity
of the crayfish. Several hours after, Ca,
Mg, Na, K, Pi and glucose level of the
hemolymph were examined. There was no
Significant difference in those minerals
between the injected group and the
control. On the other hand, glucose level
in the group which was administered with
the fraction containing larger extract in
the MW size was Significantly lower than
the control. These results seem to be
interesting considering the physiological
role of calcitonin-immunoreactive
substance in the crayfish.
EFFECTS OF ADMINISTRATION OF GOLDFISH
CALCITONIN ON THE SERUM CA CONCENTRATIONS
IN THE GOLDFISH
K. Ukawa, N. Suzuki and Y Sasayama
Dept. Gur IesL@l sy, Fac. Ge SOAs 5 Toyama
Univ., Toyama 930, Japan
It has been well known that calcito-
mins of the eel and salmon show hypocal-
cemic activity when administered to mam-
mals. Nevertheless, roles of calcitonins
in those fishes have not been clear. We
developed a new calcitonin from the gold-
fish. In the present study, using goldfish
ealcitonin, effects of administration on
the serum Ca levels in the goldfish were
Studied (Experiment I). One nmol of the
goldfish calcitonin was administered per
100 g body weight. Furthermore, we raised
anti-goldfish calcitonin polyclonal anti-
serum in guinea pigs. This antiserum was
administered to the goldfish to know whe-
ther serum Ca level is affected or not
(Experiment II). The antiserum was diluted
to 100 times, and 10 ul of which was ad-
ministered per 100 g body weight. In those
two experiments, both calcitonin and its
antiserum were administered through a can-
nula into the arterial bulb. Blood was
taken through the cannula periodically
Giulio tiger Gh loses} ayel Irons, i, untel sese(o)m ohio
chial vessels during 1 hr in Expt. II. As
a result, in both experiments, no differ-
ence in the serum Ca level was recognized
between the experimental groups and the
eontrols. These results suggest that some
conditions may be necessary for calcitonin
to express its hypocalcemic effect, and
that calcitonin level in the blood may be
so low as being negligible in the goldfish
SEXUAL DIMORPHISM AND STRAIN DIFFERENCE
IN MOUSE ANOCOCCYGEUS MUSCLE.
Y. Fukazawa, A. Suzuki, T. Iguchi, N. Takasugi and
H. A. Bern*. Dept. Biol., Yokohama City Univ.,
Yokohama; *Dept. Integrative Biol. and Cancer Res.
Lab., Univ. California, Berkeley, CA, U.S.A.
The anococcygeus muscle (AcM) is a paired, thin
sheet of smooth muscle inserting on the rectum,
having a tendinous origin largely on sacral
vertebrae. The cross-sectional area of AcM in the
juxtarectal region in 90-day-old male mice was
significantly larger than that of females in 3 strains:
BALB/cCrgl, ICR/Jcl and C57BL/Tw. The AcM
area in female mice showed a strain difference:
BALB/c > ICR>C57BL. The AcM area in 60-day-
old ICR male mice castrated at 30 days of age was
significantly smaller than in intact males, and that
in ovariectomized females was significantly larger
than in intact females. Implantation of a
testosterone or estradiol pellet (12 mg) on the day of
operation inhibited the change in AcM area. ICR
female mice exposed neonatally to diethylstilbestrol
had significantly larger AcM than controls.
Expression of androgen receptor (AR) and estrogen
receptor (ER) in AcM of 15- to 120-day-old mice was
studied immunohistochemically in ICR and C57BL
mice. The AcM in both strains showed positive AR
and ER immunostaining at 15 days. In ICR mice,
both AR and ER in AcM disappeared by 90 days.
AR and ER (male) and ER (female) were present in
the AcM of C57BL mice at 90 days. The results
suggest that androgen and estrogen play an
important role in sexual dimorphism of the mouse
AcM.
DISTRIBUTION OF EEL ATRIAL AND VENTRICULAR
NATRIURETIC PEPTIDE IN EEL HEART: ANALYSIS
BY IMMUNOCYTOCHEMISTRY USING MOLECULAR-
SPECIFIC ANTISERUM.
Y. Takeil, N. Nemoto and T. Kameya3
locean Res. Inst., Univ. Tokyo, Tokyo and
2Electronmicrosc. Lab. and 3Dep. Pathol.,
Kitasato Univ. Sch. Med., Sagamihara.
The radioimmunoassay (RIA) specific to
eel atrial or ventricular natriuretic
peptide (ANP or VNP) measured ANP almost
exclusively in eel atria, but VNP was
measured comparably both in atria and
ventricles. Immunohistochemistry using
the specific antiserum localized ANP only
in eel atria and VNP in both atria and
ventricles. However, the atrial staining
was much greater than that expected from
the concentration measured by the RIA.
This indicates some crossreaction of VNP
antiserum to ANP despite no crossreaction
in the RIA. The pretreatment of VNP
antiserum with 10-8M eel ANP eliminated
the crossreaction without affecting
crossreaction to VNP. The immunogold
electronmicroscopy using the pretreated
antiserum revealed that atrial granules
contained both ANP and VNP, whereas
ventricular granules contained only VNP.
The double-antibody method using gold
particles with different sizes showed that
ANP and VNP co-localized in the same
atrial granules.
1274 Endocrinology
UROTENSIN-LIKE IMMUNOREACTIVITY IN THE NERVOUS SYSTEM OF LOWER
VERTEBRATES AND INVERTEBRATES
§.0ka', A.Chiba’ and Y.Honma?. 'Dept.of Biol. ,Nippon Dent.Univ.,
Niigata, *Sado Mar.Biol.Stat.,Fac.of Sci.Niigata Univ. ,Niigata.
Using specific antisera raised against urotensins(U) I and II,
immunohistochemical examination was carried out on the nervous
system of some lower vertebrates and invertebrates from the
phylogenetic point of view in the caudal neurosecretory system.
Both UI- and Ul]-immunoreactivities were demonstrated in the
brains of two amphibians (Rana catesbeiana and Hynobius
sadoensis) and two primitive bony fishes (Lepidosiren paradoxa
and Polypterus senegalus), but only Ul-immunoreactivity was
found in the brain of hagfish (Paramyxin atami). In the
Polypterus spinal cord, UJI-immunoreactivities occurred in the
terminal part, whereas UJ-immunoreactivities were widely
distributed in the greater part of cord excepting for terminal
region. No immunoreactivities were seen in the spinal cord of
Hinobius and Paramyxine, but Ul-immunoreactivities were detected
in the nerve cord of amphioxus (Branchiostoma floridae) and in
the cerebral ganglia of the crab (Gaetice depressus) the squid
(Todarodes pacificus) and the sea hare (Aplysia kurodai). These
results suggest a long history of the caudal neurosecretory
peptides, especially UI or its related molecules, in association
with the evolution of the central nervous system.
CO-LOCALIZATION OF LH& AND PROTEOGLYCAN-—
LIKE IMMUNOREACTIVITY IN THE PITUITARY OF
MALE AND FEMALE RATS.
N.Hama , A.Sato“, M.Nishizyka’, T.Hari-
gaya, N.Ooshima“, and Y.Arai Sch. Agr.,
Meiji Univ., Kawasaki, “Dept. Biomol. Sci.,
Fac. Sci., Toho Univ., Narashino, Dept.
Anat., Juntendo Univ. Sch. Med., Tokyo.
The anterior pituitary of adult female
rats contained cells that were immunocyto-
chemically stained with an monoclonal
antibody raised against proteoglycans (PGs)
in the brain of rats. Since ultrastructure
of PG-like immunoreactive (irPG) cells
resembled that of gonadotropin (GTH) cells,
co-localization of leteinizing hormone
(LHB) and irPG was studied. All of the
irPG cells were LHB-immunoreactive in adult
female rats, whereas some were immunonega-—
tive to the anti-PG. These results showed
that irPG cells were a population of GTH
cells. During the estrous cycle, frequency
of co-localization, namely, irPG-LHB cells
per LH®B cells, varied, being greater at
proestrus. As for adult male rats, irPG
was not seen in the anterior pituitary. By
contrast, in castrated male rats that were
treated with estradiol-178 for 2 weeks,
irPG cells were numerous. Most of these
cells were LHB-positive, some being LHUB-
immunonegative. These finings indicate
that estrogen increased the PG-like immu-
noreactivity in the anterior pituitary.
The irPG-substance in the pituitary may be
involved in the reproduction.
DISTRIBUTION OF PROTEOGLYCAN-LIKE IMMUNORE-
ACTIVITY IN THE ANTERIOR PITUITARY OF MALE
AND FEMALE RATS.
A.Satol, N-Hama“, M.Nishizuka, N.Ooshimal,
T.Harigaya“~, and Y.Arai~. ~Dept. Biomol.
Cie.) hac. (Ser... Lonos Unwin REPRE OLD |
Sch. Agr., Meiji Univ., Kawasaki, & ~Dept.
Anat., Juntendo Univ. Sch. Med., Tokyo.
Distribution of the immunoreactivity
specific to an anti-proteoglycan (PG)
monoclonal antibody, raised against rat
brain PGs, in the anterior pituitary of
rats was studied. In adult female rats,
PG-like immunoreactivity (irPG) was seen in
the cytoplasm of glandular cells. By
contrast, irPG were not seen in adult male
rats. Similarly, irPG was seen in the
female on postnatal day 41, but not in the
male. However, irPG were seen in both male
and female rats on postnatal day 5 and 15.
These results indicate that appearance of
the irPG in the anterior pituitary was
sexually different. However, existence of
the irPG was inherently programmed in both
the male and female rats. The appearance
seemed suppressed in adult male rats.
Castration for 2 weeks and treatment
with testosterone following to castration
decreased the irPG in females. By con-
trast, castrated male rats that were treat-
ed with estradiol-178 for 2 weeks had
numerous and intensely stained irPG-cells.
These findings indicate that the appearance
of the irPG depends on estrogen. rt re-
mains unsolved whether androgen suppressed
the appearance the irPG.
Cell Biology and Morphology 1275
INTERSPECIFIC TRANSPLANTATION OF GERMINAL
MICRONUCLEUS IN PARAMECIUM
K. Oka and K. Mikami. Res. Inst. for Sci.
Educ., Miyagi Univ. of Educ., Sendai.
Ciliates have two types of nucleus, the
somatic macronucleus (mac) and the germinal
micronucleus (mic). To investigate a
relationship between species belonging to a
genus Paramecium, the mics were transplanted
into cells of other species.
In the present work, amicronucleate cells
of P. caudatum were transplanted with the
mic of P. trichium. The transplanted mic
was retained in the cytoplasm for a while.
In the cytoplasm, some of the mics apparent-
ly divided. However, the mic division did
not couple with cytokinesis so that the re-
nucleated clone contained amicronucleates,
uni-micronucleates and two or more micro-
nucleated cells. Whereas the shape of the
mic of P. trichium is spherical before
transplantation, the transplanted mic some-
times looked abnormal; spindle or rod shape.
Amicronucleate cells grow very slowly
after mic removal. These amicronucleate
cells recover its fission rate after
reimplantation of P. caudatum mic. However,
they did not recover the rate after im-
plantation of P. trichium mic. The result
shows that the mic of P. trichium did not
perform its function in the cytoplasm of P.
caudatum.
When ordinary micronucleate cells of P.
caudatum was transplanted with the mic of P.
trichium, the behavior of the implanted mic
was almost the same as that mentioned above.
MICRONUCLEAR DIVISIONS AFTER MACRONUCLEAR
REMOVAL DURING CONJUGATION OF PARAMECIUM.
K. Mikami. Res. Inst. for Sci. Educ.,
Miyagi University of Education, Sendai.
Each cell of Paramecium caudatum has a
germinal micronucleus (mic) and a somatic
macronucleus (mac). When the macs were
removed from both cells of a conjugating
pair before metaphase of of the lst meiotic
division, the mics did not divide. When the
Macs were removed at the metaphase or the
anaphase, however, the mic repeated division
so that it produced more number of nuclei
than usual 4. In an extreme case, a mic
divided 4 times and produced 16 nuclei. The
result shows that the mic is able to repeat
division in the absence of the mac.
To know whether the mac is necessary for
mic division during the postzygotic process,
the mac was removed at the stage of syn-
karyon or two postzygotic nuclei. Then, mic
(synkaryon) divided twice or more times. In
an extreme case, 32 nuclei were produced.
These results show the mic is able to
divide in the absence of the mac after meta-—
phase of the 1st meiotic division and lead
to the following hypotheses: the informa-
tinal factors required for mic division have
been prepared prior to the mac removal, or
the factors are prepared by the mic itself.
When cells were treated with Actinomycin
D (100ug/m1) at the metaphase or postzygotic
stage, mic division was inhibited in most of
the cells. Although the results seem to
support rather the later hypothesis, so far,
we cannot exclude the former hypothesis.
BEHAVIOR OF MICRONUCLEI TRANSPLANTED
BETWEEN THE TWO DIFFERENT EUPLOTES DURING
CONJUGATION.
K.Sato. Dept. of Biol., Naruto Univ. of
Educ., Naruto.
A study in which micronuclear
transplantation was performed reciprocally
between the two different species of
Euplotes (E. octocarinatus, E. patella)
has been reported (Proceedings of the 62
Annual Meeting of the Zoological Society
of Japan, 1991). In the stock derived
from the operated E. octocarinatus cells
into which E. patella micronuclei were
transplanted (called Tmp(K18)-0(1-17)9),
alive progenies from this 60 homotypic
conjugants was not obtained, but spherical
macronuclear anlagen developed. Also ina
stock of the opposite transplantation
combination, called Tmo(1-14)-p(K18)12, 56
exconjugants were isolated, but they died
out. Though meiosis, eight nuclei stage
after meiosis and small swelling
macronuclear anlagen were observed, extra
nuclei were also observed in the
exconjugants. From results, the
transplanted micronuclei derived from the
different species can undergo meiosis and
other nuclear divisions in the cells of
the different species, although
macronuclear anlagen could not develop.
If the informations for meiosis and the
other nuclear behavior are produced by
macronuclei, the informations seem to
resemble closely between the two Euplotes.
ELECTRON MICROSCOPY ON MEIOSIS OF MICRO-
NUCLEUS IN Tetrahymena.
Y. Suganuma. Biol. Lab., Narasahojogakuin College, Nara.
Mitosis of the micronucleus in Tetrahymena thermophila is
a "closed acentric" type in which no centriole exists and the
nuclear membrane progresses without breakdown. In first,
second and third prezygotic divisions, chromatin loosen into
thin threads and loosely congregate to form chromosomes with
low density of electrons. Cap-shaped kinetochores can be
found in the nucleus during its division, which consist of a core
with high electron density (external diameter, 210 nm;
thickness, 20 nm) and a halo of low electron density around the
core (thickness, 20 nm). The chromosomes are attached to the
inside of the core. Microtubules (KM) are attached to the halo,
which show polar alignment. The other side of the KM ends at
the polar region of the nucleus. As the KM becomes shortened,
chromosomes separate and shift in both polar directions. During
anaphase of the third prezygotic division, a part of the
elongated nucleus nearest to the cell-to-cell junction is
positioned parallel to the junction area. This part of the nucleus
remains within the junction area even after the division and
becomes a migratory pronucleus. On the other hand, the other
part of the nucleus which has elongated towards the
macronucleus approaches the joint region after the division,
becoming a stationary pronucleus. Chromatins within both the
pronuclei are thick and short with high electron density and are
scattered within the nuclei. However, when a part of the
migratory nucleus migrates into the partner cell, the chromatins
immediately loosen and become thinner. After migration, the
chromatin becomes a loose configuration like chromosomes.
1276 Cell Biology and Morphology
ULTRASTRUCTURAL COMPARISON OF PARORAL REGION
OF AMICRONUCLEATE AND MICRONUCLEATE CELLS DURING
CONJUGATION IN PARAMECIUM TETRAURELTA.
Y. Yashima. Dept. of Biol., Sch. of Lib. Arts
and Sci., Iwate Med. Univ., Morioka.
When the meiosis was completed during
conjugation in Paramecium tetraurelia, 8
meiotic products were observed and only one of
them located in the paroral region survived.
In order to understand this mechanism, the
morphology of the paroral region and that
surrounding the nuclei of the conjugants which
were present between the stage of nuclear
moving into the paroral region and nuclear
exchange were examined by electron microscopy.
A meshwork structure composed of microtubules
was present in the paroral region of the
conjugant that consisted of 3 types of
combinations with amicronucleate cells (A cell)
and micronucleate cells (M cell) in the stage
prior to the nuclear migration into the partner
cell. Moreover, microtubules were present around
the micronucleus located outside the paroral
region in the telophase of the secondary
meitotic nuclear division. At the stage of
micronuclear exchange, various kinds of vesicles
in size and shape, i.e.,rough endoplasmic
reticulum, discoidal vesicles, golgi apparatus,
coated vesicles were present directly behind
the migratory nucleus in the paroral region of
both the A cells and the M Cells.
These results suggest that the formation of
the meshwork structure may be controlled by
the macronucleus,and the nuclear migration
into the paroral region may be controlled by
microtubules.
DISTRIBUTION AND PROLIFERATION OF CORTICAL
UNITS IN THE CILIATE PARAMECIUM TRICHIUM.
M. Okubo, T. Kosaka and T. Takahashi.
HOO IL ANS Bo 5 CK Cat Seals, bhiicoglniyma
Univ., Higashi-Hiroshima.
The cell surface of Paramecium is cov-
ered with many cortical units longitudi-
nally arranged in kineties. In these units
main components are cilium(c), basal
body(bb), kinetodesmal fiber(kd) and
lattice. Each unit of the morphostatic
cell may contain one or two bb. It, howev-
er, has not been cleared yet whether all
of them are ciliated or not. The purpose
of this work is to examine the detail
morphology, cytogeometrical distribution
and proliferation processes of the units
by silver impregnation technique and by
scanning electron microscopy. According
to the number of contained bb and c, the
cortical units could be classified the
following three types which distributed in
the specifically localized field of the
cell; 2bb-2c unit in the ventral left
anterior field, 2bb-1c unit in the field
nearby posterior pole of the cell, and
1bb-1c unit in the rest field. Moreover,
the mixed field of 2bb-1lc and ibb-1ec units
was also observed in the region near the
border of their fields. These cortical
units proliferated in all over the cell
surface during cell division except for
the certain parts of the 2bb-2c unit and
1bb-1lc unit fields. The present work
indicated that there were four modes in
the proliferation processes of the units.
ORAL ASSEMBLY IN THE .CILIATE GLAUCOMA
SCINTILLANS.
M.Suhamal and K.Matsumoto2. 1Fac. of
Liberal Arts, Shikokugakuin Univ.,
Zentsuji and 2Zool. Inst., Fac. of Sci.,
Hiroshima Univ., Higashihiroshima.
We have investigated the assembly and
alignment of basal bodies during develop-
ment of oral apparatus(OA) in the right-
(RH) and left-handed(LH) cells of Glaucoma
scintillans by SEM observation. Observa-
tion was done mainly on isolated OAs and
peeled pellicles with anlage of OA. At the
lst step of fromation of membranelles basal
bodies arranged in a row at the left edge
of rudimentary field were commonly seen in
RH cells, and then each basal body became
pair, adding a basal body to its left side
(cell's). The 3rd-7th rows of basal bodies
were orderly added to the left side. The
basal body displacement occurred at the
anterior ends of 3 membranelles(Ml, M2, M3)
during oral development in RH cells; at the
anterior tip of M2, 6 basal bodies moved
toward M3 and made a peculiar sculptured
appearance, while the sculptures of Ml and
M3 were not intricate in appearance. The
sculpture of M2 in many LH cells was found
at the posterior end, indicating 180 rota-
tion of M2. Cilia grew from the anterior
end of eacn membranelles in RH and LH cells
regardless of the direction of alignment of
basal bodies. The bundles of membranellar
basal microtubules connecting between Ml,
M2 and M3 were found at the late period of
oral development.
REGENERATION OF LONGITUDINAL UNEQUALLY
SECTIONED FRAGMENTS IN THE STICHOTRICH
CILIATE ONYCHODROMUS QUADORICORNUTUS.
T. Sato, T. Kosaka and T. Takahashi
Zool.inst., Fac. of Sci., Hinroshimasunive.
Higashi-Hiroshima.
For interpretation of pattern formation
in ciliate, Frankel(1989) proposed an
application of the cylindrical coordinate
model(CCM) which has been developed from
polar coordinate model for patterning.
According to this model, it is considered
that if one-half or more of the cell cir-
cumference is removed, the fragment might
undergo mirror-image duplication of the
surface structures in the regeneration.
The problem, however, as to whether the
presumption is correct has been remained
to examine. We, therefore, examined the
morphogenetic fate of longitudinal un-
equally sectioned fragments (right:left =
2:1) of the large stichotrich edaiace
Onychodromus (max. ca. 600um in length).
About 70% of right large fragments and
about 15% of left small ones regenerated
to normal cells within 2 days after the
operation. All of the other fragments did
not regenerate and died; that is, mirror-
image duplication were not observed in the
present operations. These findings indi-
cated that it may difficult to application
of the CCM hypothesis to the pattern
formation in the regeneration of the
longitudinal unequally sectioned fragments
of this ciliate.
Cell Biology and Morphology 1277
IMMUNOLOGICAL EXAMINATION OF THE CYST
WALL SPECIFIC PEPTIDES IN A STICHOTRICH
CILIATE, HISTRICULUS CAVICOLA.
M. Himura and T. Matsusaka. Dept. of Biol. Sci., Fac. of
Sci., Kumamoto Univ., Kumamoto.
A rabbit antiserum raised against isolated cyst wall of a
ciliate, Histriculus cavicola specifically decorates, immuno-
electron microscopically, the 3 layered cyst wall, ecto- meso-
and endocyst, and also autophagic vacuoles. Several peptide
bands was recognized by the antiserum on the Western blot of
the cyst wall. Especially, 4 of these peptide bands, 180, 54,
48, and 37 kD, showed intense reaction to the antiserum. As
the 180 kD peptide has been known to locate in the endocyst,
antibodies against the other 3 peptides were affinity purified
from the immuno-blots to determine the localization of these 3
peptides within the cyst. Affinity purified antibodies against 54
kD peptide cross-reacted with the 48 and the 37 kD peptides
and decorated the ecto- and the mesocyst. The same results
were obtained from the antibodies purified from 48 and 37 kD
peptides. To determine possible involvement of specific sugar
residues or polysaccharides in the cross-reaction, Western blot
filters were exposed to 4 lectins, Con A, WGA, PNA, and
DBA and were also examined by PAS-test. Only 54 kD
peptide was recognized by DBA, indicating the involvemen of
N-acetyl galactosamine in the peptide. The other 2 peptides,
however, were neither recognized by the lectins tested nor
reacted to PAS-test. These results suggest that sugar residues
are not involved in the cross reaction. The results of partial
proteolysis of the 3 peptides using protease V8 indicated that
given 2 peptides out of the 3 contained a few common peptide
fragments but that no common fragment among 3 peptides were
present. Some of these fragments were recognized by the
antiserum. These results may indicate that the 3 peptides may
contain common epitopes, and that the common epitopes may
be peptides.
ANALYSIS OF SPECIFIC ANTIGENS IN ACAN-
THAMOEBA BY MONOCLONAL ANTIBODIES.
Y. Hirukawa!, H.Ohba?., J. Chiba? & T. Tsuruhara!.
'Dept.Biol., fokyo Gakugei Univ.,Koganei and
2Dept. Biol. Sci. & Tech., Sci. Univ. of Tokyo, Noda.
We have developed hybridoma cell lines which
secrete murine monoclonal antibodies to the cysts and
trophozoites of Acanthamoeba polyphaga (ATCC
30871, Page 3a). The cross-reactivity tests of the
monoclonal antibodies were performed by using
other Acanthamoeba strains (A culbertsoni, A. astro-
nyxis & A.castellanii) in enzyme-linked immuno-
sorbent assay and immunoblotting experiments.
These analyses revealed that the monoclonal anti-
bodies recognized primarily the polypeptides of an
comparative low molecular weight of homologous
amoeba. And we also observed the binding mono-
clonal antibodies to the cyst and trophozoites of the
same strain by an indirect immunoperoxidase
technique for light microscopy. Further biochemical
and morphological works are in progress to study the
characteristics especially in cell differentiation.
PHYSIOLOGICAL AND MORPHOLOGICAL
CHARACTERIZATIONS OF ACANTHAMOEBA
HATCHETTI-ISOLATED FROM SOIL SAMPLES.
K. Watanabe!, H. Horikami*, K. Ishii? and
T. Tsuruhara1!.
Dept. Biol., Tokyo Gakugei Univ., Koganei and
2Lab. Biol., Hosei Univ., Tokyo.
Acanthamoeba species are a small free-living
amoebae and are found very widely in nature and 18
different established species are now described, but
Acanthamoeba hatchetti is known only from
brackish-water sediments in USA. We have isolated
two strains of Acanthamoeba hatchetti from soil
samples taken in Tokyo and Naha, and have
successfully established pure culture lines starting
from single cysts. These strains are able to grow and
encyst on agar plates containing artificial seawater
(salinity: 30 %o). Their cysts are small and refractile
with thick ectocyst and have the three- or four-
endocyst arms (mean cyst diameter:11.44.m) which
gives a triangular shape. We have now investigated
their patterns of isoenzymes to compare with other
strains.
DISINFECTION AGAINST ACANTHAMOEBA’S CYST FROM HU-
MAN KERATITIS.
H. Horikami!, K. Ishii!, H. Yamaura?, and Y. Ishibashi?. 1 Lab. of Biol.,
Hosei Univ., Tokyo, 2 Dep. of Parasitol., Tokyo Women’s Med. Coll., To-
kyo, 3 Dep. of Ophthalmol., Tsukuba Univ., Tsukuba-shi.
The cysticidal properties of various test solutions were examined against 2
strains of Acanthamoeba isolated from keratitis. The cysts(2-3 weeks old,
10° cells) of A. polyphaga, strain Fuks and A. castellanii, strain Toks were
soaked overnight into 1.5 ml of test soln in plastic microtube at room temper-
ature, and then washed 4 times by centrifugation (600Xg/3 min) with amoeba
saline. The sediment was inoculated on non-nutrient agar plate smeared
with 5 % yeast extract and 10 % glucose and incubated at 30°C for 10 days.
The plates were examined daily for amoebic growth.
1) 0.2% Osvan soln(DAIGO), 30 % ethanol, 1 % commercial sodium hy-
pochlorite soln "HAITER”(KAO CORP.), 0.1 % sodium hypochlorite soln
“Antiformin”"(WAKO CHEMICALS), 1 N NaOH soln, 0.1 % thymol soln
and 1 % picric acid soln inhibited perfectly the excystment, especially 4 %
HAITER and 10 % Antiformin induced cytolysis, but the double cyst walls
were maintained. On the other hand, 50 % glycerin soln, 1 N HCI soln, 1 M
soln of LiCl , NiCl, BaCl5, ZnCl,, and MgCl, were not effective.
2) In spite of treatment of cysts with 0.1 % soln of HAITER or 0.05 % soln
of Antiformin was not able to inhibit the excystment, addition of 10 % of
glucose, sucrose or mannnitol into the test soln inhibited perfectly the excyst-
ment This phenomenon could not induced with yeast extract or amino acid.
The cysticidal action of chlorine on Acanthamoeba was reported by Jonck-
heere and Voorde(1976). In comparison with their chlorine content, our re-
Sults show that the content was at least 100 times lower with the addition of a
sugar, and this new disinfectant might be more safety.
1278 Cell Biology and Morphology
FOOD VACUOLE FORMATION IN PARAMECIUM FED
WITH STREPTOBACILLUS.
S.Mishima. Biol.Lab., Coll.Gen.Educ.,
Ibaraki Univ., Mito.
Previously we reported that the food
vacuoles produced by Paramecium fed with
streptobacillus(SB) were not spherical but
they had a strange shape such as a string
of cells or a bundle packing the strings.
Further it was found that Paramecium pro-
duced spherical food vacuoles including SB
when mono- or oligo-saccharide was added
to the culture. In the present study we
examined the shape of food vacuoles when
added with a mixture of SB and polystyrene
latex particles. Formation of abnormal
vacuoles(10-15%) such as apparently spher-
ical ones with long-ranged cells sticking
out of their surface or vacuoles peaked
at both longitudinal ends like an American
football were observed. The SB prepara-
tion contained long strings of more than
6 cells at the proportion of 10%. Since
the diameter of an ordinary food vacuole
corresponds to the length of a string 4
cells, it is supposed that formation of
these abnormal vacuoles might occur when
a vacuole incorporates longer SB than the
diameter of a vacuole, whereas Paramecium
given saccharide can produce a spherical
vacuole by incorporating them curved along
its half circumference even in the case of
long SB.
EFFECTS OF HEAT-SHOCK ON REGENERATION IN THE
CILIATE PSEUDOUROSTYLA LEVIS: SDS-PAGE ANALYSIS
OF THE PROTEINS IN THE REGENERATING AMICRO-
AND MICRONUCLEAR FRAGMENTS
T. Takahashi. Zool. Inst., Fac. of Sci., Hiroshima
Univ., Higashi-Hiroshima.
For analysis of the somatic functions of
micronuclei in the multinuclear ciliate Pseudo-
urostyla, effects of heat-shock on regeneration
of amicro- (ami) and micronuclear (ml) fragments
were examined. At 2h after the cell division
many Gl cells were transected into two frag-
ments at posterior to the mouth, and then they
were maintained at 23 C. At 1.5h after the
operation these fragments were treated at 38.5 C
for 20min. Subsequently they were fixed for
silver impregnation with protargol technique, or
were transferred to sample buffers for SDS-PAGE
at constant intervals of lh. About 300 frag-
ments per lane were needed to prepare for the
SDS-PAGE samples. Regeneration of non-heat-
shocked ami and ml fragments completed about 5-
6h after the operation. In contrast, completion
of regeneration delayed for about 3-4h In heat-
shocked both ami and mi fragments. SDS-PAGE
indicated that each of lanes contained more
than 50 bands, but any specific proteins for the
mouth part formation could not be determined
Moreover, about 8KD protein was always observed
as major band In mi fragments, but faintly In
ami fragments. The problem as to whether this
difference {is related to the somatic functions
of micronucle! remains to be analyzed in the
future.
RESTORATION OF THE REDUCED FISSION
RATE OF THE JUMYO MUTANT OF PARAME-
CIUM TETRAURELIA.
Y. Tokusumi!, H. Fujisawa!, Y. Takagi! and K. Kaji2
'Dept. of Biol., Nara Women's Univ., Nara,
2Tokyo Metropol. Inst. Gerontol., Tokyo.
The jumyo mutant of Paramecium tetraurelia has
been used for the assay of mitogenic substances by the
restoration of its reduced fission rate in daily reisola-
tion culture. We have shown that 2% or more fetal
bovine serum (FBS) was mitogenic for the jumyo mu-
tant, as well as the cell-free conditioned medium of a
variety of Paramecium cultures. We here tested for the
effect of mammalian growth factors (TGFa, EGF,
PDGF, IGF, b-FGF) and the cell-free conditioned
medium of Tetrahymena. TGFa and EGF were some-
what effective at very high concentrations (50 ~ 300
ng/ml), although no growth factors were effective at
20 ng/ml. The cell-free fluid of T. pyriformis and T.
thermophila concentrated 100-fold by ultrafiltration
was also effective. When wild type cells were used,
their nutritionally reduced fission rates were restored
by a concentrate of culture medium whether or not it
included cells of Paramecium or Tetrahymena, but not
restored by FBS. There is yet much to do before we
conclude decisively, because the fission rate of the
jJumyo mutant is such unstable as to become suddenly
high without any mitogens.
FUNCTION OF TETRAHYMENA EXTRACT FOR
INDUCTION OF MATURATION OF XENOPUS
OOCYTES
E.Ogawa!, M.Fujishima!, M.Yamashita?,
Y.Nagahama? and N.sagata?. 1Biol. Inst.,
Fac. of Sci., Yamaguchi Univ., Yamaguchi,
2Lab. of Reprod. Biol., Natl. Inst. for
Basic Biol., Okazaki, and 3Div. of Mol.
Genet., Inst. of Life Sci., Kurume Univ.,
Kurume.
Reinitiation of meiosis (maturation) of
Xenopus oocyte can be induced if
Tetrahymena extract is injected into
them. The activity of this meiosis-
reinitiation-inducing factor (MRIF)
differed from M-phase-promoting factor
(MPF), because action for induction of
germinal vesicle breakdown (GVBD) of MRIF
needed the recipient oocyte protein
synthesis. However, MPF-activity appeared
in the MRIF-injected oocytes. Immunoblots
using antibodies for Mos protein and
PSTAIR sequence of cdc2 showed that MRIF
induced appearance of Mos and
dephosphorylation of cdec2 in the oocytes
in the presence of the oocyte protein
synthesis. This result suggests that
primary function of MRIF in the oocyte is
induction of Mos protein, and then the Mos
induces dephosphorylation of cde2 to
induce active-MPF and GVBD.
Cell Biology and Morphology 1279
INBREEDING OF THE CILIATE PARAMECIUM
BURSARIA.
S. Matsuda, T. Kosaka and T. Takahashi.
Zool. Inst., Fac. of Sci., Hiroshima
Univ., Higashi-Hiroshima.
Two breeding systems, inbreeding and
outbreeding, are known in ciliates. This
study was made to examine how these breed-
ing systems function as a sexual reproduc-
tive strategy in a natural population of
ciliates. Using Paramecium bursaria
syngen 1, which is considered a typical
outbreeding species, the effect of in-
breeding (sib-cross) on the viability of
exconjugants was studied. Four Fl clones
belonging to different mating types (I,
II, III and IV) were used. They originat-
ed from a cross between parental stocks
OK-312(1) and OK-223(IV). When any two
complementary mating type II, III or IV
were mixed together, the viability of the
exconjugant cells (F2) was over 90%.
However, when the mating type I clone was
used in crosses with any of the other
mating types, the viability of exconjugant
cells was under 75%. The cause of the low
viability remains unclear. The viability
of outcrossing (conjugation between two
stocks originated from different places)
was generally high. Thus, the results
show that with occasional exception in-
breeding as well as outbreeding could work
to maintain viable populations of P.
bursaria in nature.
A BIOLOGICAL CLOCK SYSTEM OF MATING TYPE
REVERSALS RHYTHM IN PARAMECIUM
MULTIMICRONUCLEATUM, SYNGEN 2.
S. Kamiya! and I. Miwa’. ‘Dept. of Biology, Fac. of Sci.,
Ibaraki Univ. *Biol. Lab., Coll. Gen. Educ., Ibaraki Univ. ,
Mito.
Cells of P mulomicronuclentum change the mating type two
times in a day, they express the mating type III for a part of a
day and the complementary mating type IV for the remainder of
the day. Cells of a stock used in this experiment changed the
mating type from III to IV about 11 hours after the onset of the
dark period when they were put in the various light and dark
cycles. The length of the period of mating type IV depended on
the dark period. Therefore their timekeeping system is
supposed to be a clock of sandglass type, which begins to time
with the signal at onset of darkness. But the mating type
reversals rhythm was continued under the both conditions of
constant light and darkness. Thus, an endogenous circadian
oscillator also controls the rhythm of mating type reversals in P
multimicronucleatum.
In addition, according to a result of an experiment of low
temperature treatment, it became clear that they needed a certain
reaction during the light period in order to change the mating
type from III to IV. Ina light pulse experiments, the timing of
mating type transition was delayed when they were exposed to
a light pulse at the first half of the dark period, but it was not
delayed when they were exposed to a light pulse at the latter
half of the dark period.
Now we are continuing to investigate the effect of many
drugs of known specificity on mating type reversals of P
multimicronucleatum.
EARLY MATURE MUTANT ON THE DEVELOPMENTAL
CLOCK IN PARAMECIUM BURSARIA
I. Miwa!, S. Takaya? and Y. Tomioka®. Biol. Lab., !Coll.
Gen. Educ., *Fac. of Sci., 9Fac. of Edu., Ibaraki Univ., Mito
An exconjugant clone of Paramecium has a well-defined life
cycle, consisting of the period of immaturity, maturity and
senility. During the immaturity period, Paramecium is unable to
mate even under appropriate conditions. The duration of the
immaturity period can be measured by the developmental clock
which depends on the number of fissions after conjugation.
The exconjugant clones of P bursaria show adolescence more
30 fissions after conjugation during in the transition from
immaturity to maturity, in which they express the one of two
genes determined mating type.
We isolated recently a early mature clone after treated with 2
pg/ml nitrosoguanidine (MNNG) for 5 hours. This clone
exhibited mating reactivity 15-20 fissions after conjugation, and
genetic control of the early mature character was shown by
Mendelian segregation. Homozygote of the early mature gene
exhibited mating ability 8-12 fissions after conjugation.
Though the length of adolescence in wild type stocks was 12-
14 fissions, that of the early mature mutant was shortened was
2-4 fissions.
P. bursaria exhibits circadian rhythm of mating reactivity.
Then we investigated the correlation of both clocks of
development and circadian. The stocks shown short period
ciradian rhythm of photoaccumulation became to mature earlier
than the stocks shown long period of circadian rhythm. But
this early mature mutant showed almost the same circadian
period as the wild type stocks shown short period.
COLD RESISTANCY OF SOME CNR MUTANTS IN
PARAMECIUM CAUDATUM.
K.Hiwatashi. Dept. of Biotech., Senshu
Univ. of Ishinomaki, Ishinomaki, Miyagi.
We have reported (Hiwatashi & Skoblo,
1991) a CNR mutant discovered in a natural
stock of Paramecium caudatum collected in
Russia. Since CNR mutants are defective
in a vital function of paramecium cells,
the discovery of natural stock containing
a CNR gene suggests that the CNR gene may
in some respect give a selective advantage
for the stocks containing it. Resistancy
to low temperature was tested for the
Russian CNR stock, since the stock was
collected in a cold district. Five differ-
ent CNR mutants, cnrA, cnrB, cnrD, cnrD
and the Russian CNR were cultured in daily
isolation lines at three different temper-
ratures, 5°, 10° and 25°C. At the temper-
ratures of 10° and 25°C, no remarkable
difference was seen among stocks of wild
type, CNRs and their Fl heterozygotes, but
at 5°C, stocks of cnrC€ and the Russian CNR
showed a significant cold resistancy com-
pared with other CNR stocks. However,
since a few wild-type stocks and the stock
of cnrC homozygote expressing wild type
also showed the cold resistancy, the cold
resistancy of the above resistant CNR
stocks seems not to be controlled directly
by the CNR genes involved.
1280 Cell Biology and Morphology
PRODUCTION OF MONOCLONAL ANTIBODIES
SPECIFIC FOR HOLOSPORA OBTUSA OF
PARAMECIUM CAUDATUM.
H.Dohra and M.Fujishima. Biological
Institute, Fuculty of Science, Yamaguchi
University, Yamaguchi.
The gram negative bacterium H.obtusa
is a macronuclear specific symbiont of the
ciliate P.caudatum. This bacterium changes
its morphology in good accordance with the
growth of the host cells; reproductive
short form and infectious long form. When
the host cells starve in the stationary
phase of the growth, the reproductive
short forms cease binary fissions,
elongate themselves and differentiate the
infectious long forms. To detect bacterial
stage-specific substances, in the present
study, we intended to get monoclonal
antibodies(mAbs) specific for the
infectious long forms.
We isolated infectious long forms and
their whole cells, sonicated brei and
purified proteins from 2D-SDS- and native-
PAGE gels of the infectious long forms
were injected into mice as the antigens.
As a result, seven mAbs including
infectious form specific ones were
obtained. We show intra-cellular
localities and molecular weights of the
antigens by indirect immunofluorescences
and immunoblots.
DIFFERENTIATION FROM REPRODUCTIVE FORM TO
INFECTIOUS FORM OF ENDOSYMBITOTIC BACTERIUM
HOLOSPORA OF PARAMECIUM IS INDUCED BY
INHIBITION OF THE HOST PROTEIN SYNTHESIS.
M. Fujishima and N. Hirakawa. Biol. Inst.,
Fac. of Sci., Yamaguchi Univ., Yamaguchi.
Reproductive short form (1.5 ~m in
length) of an endonuclear symbiont
Holospora obtusa of Paramecium caudatum
grows by binary fissions in the host
macronucleus when the host grows. When
the host starves, however, the bacterium
also ceases the fission and differentiates
into the infectious long form (13 ~im in
length).
Short forms-bearing paramecia in the
log phase of growth were suspended in
different mediums at 25°C: (1) culture
medium, (2) phosphate-buffered saline and
(3) culture medium-containing 1 ug/ml
emetine, and then their bacterial
morphology was observed. In (1), no
bacterial elongation was induced. In (2)
and (3), the elongation began at 48 h. In
(3), the elongation was enhanced more than
that in (2), notwithstanding that the
hosts were not starved. These results
suggest that induction of the
differentiation of the infectious form by
the host starvation may be due to the
deterioration of the host protein
synthesis.
REESTABLISHMENT OF SYMBIOTIC ASSOCIATION
BETWEEN ALGAE-FREE PARAMECIUM BURUSARTA
AND SYMBIOTIC ALGAE FROM GREEN PARAMECIA.
K. Kimura, T. Kosaka, and T. Takahashi
Zool. Inst., Fac. of Zool., Hiroshima
Univ., Higashi-Hiroshima.
The incubation of algae-free(AF) Parame-
cium bursaria with symbiotic algae(SA)
from green paramecia results in the rees-
tablishment of symbiotic association of
them. The mechanism of this event, howev-
er, is hardly clear. Recently, we found
that SA remarkably changed their morpho-
logical feature with the growth of host
paramecia. In this work, therefore,
examined the relationship between the
reestablishment of symbiotic association
and the growth phase of host paramecia.
When the log and stationary phase AF
paramecia were incubated with SA from log
growing green paramecia, more than 95% and
about 60% of AF paramecia were reinfected
with the symbiotic algae. On one hand,
when the SA from stationary phase green
paramecia were mixed with log and station-
ary phase AF paramecia, successful infec-
tion rate was about 10% and 20%, respec-
tively. It has been also found in this
work that the SA can be cultured with CA
medium. When the cultured SA were incu-
bated with log and stationary AF parame-
cia, rates of reestablishment of symbiotic
association were about 90% and 50%. These
findings suggest that the reinfection
ability of symbiotic algae may change with
the growth phase of the host.
SYMBIOTIC CELLULOSE DIGESTION SYSTEM IN
THE LOWER TERMITE: ROLE OF BACTERIA.
I. Yamaoka and R. Murakami. Biol. Inst.,
Fac. of Sci., Yamaguchi Univ., Yamaguchi.
It has been clear that the cellulose
digestive system in the lower termite
consists of the complicated relation
between host and the symbionts. Especial-
ly bacteria play an important role.
One of them bacteria make the anaerobic
condition of the hindgut lumen for intes-
tinal protozoa and the other supply the
metabolites to the symbionts. In here it
suggests that one of the metabolites of
the intestinal protozoa H gasses are
reduced into CH gasses by the methanoge-
nic bacteria in the hindgut. Their bacte-
ria adhere to the surface of the hindgut
epithelium and live in some species of
the intestinal flagellates. They are
examined by the fluorescence microscope
and the electron microscope.
Cell Biology and Morphology 1281
THE ALIMENTAL CANAL IN THE HIGHER
TERMITES: ULTRASTRUCTURE OF THE MIDGUT
AND THE MIXED SEGMENT.
G. Tokuda, R. Murakami and I. Yamaoka.
Biol. MInst., Fac. of Sci., Yamaguchi
Univ., Yamaguchi.
The alimental canal of the higher
termite ( Nastitermes takasagoensis and
Odontotermes forsanus ) were examined by
the electron microscope. The midgut
epithelia in both species were similar
in the fine structure and consisted of
columnar cells and regenerative cells
formed a mass. In the columner cells the
nucleus situated at the cell apex (a side
of the lumen ) and invaginations of the
cell membrane observed at the cell base (
a side of basal lamina ). Large and with
the well developed crista mitochondria
were observed into the cell invaginations.
Large phagosome was observed in the
cytoplasm of each cell. The mixed segment
observed in the N. takasagoensis was not
observed in the O. formosanus. Another
difference of the fine structure of both
species was also clear.
SYMBIOSIS BETWEEN AN EOLID NUDIBRANCH
CUTHONA SP. AND A HYDROZOA AGLAOPHENIA
CUPRESSINA LAMOUROUX
T. Yamasu, Dept. of Biology, Div. of Gen. Educ. Univ. of the
Ryukyus, Nishihara, Okinawa.
Living specimens of an eolid nudibranch, Cuthona sp. were
collected on the colonies of a thecate and zooxanthellated
Hydrozoa, Aglaophenia cupressina Lamouroux found abundant
all the year round on patch reefs at Cape Zanpa in Okinawa
Island. The colonies of the hydroid collected were cut into small
size and reared in containers (30 cm in diameter and 12 cm in
depth, together with 2 litter of sea water) kept aerated under
fluorescent lump illumination (1500 lux) in the laboratory for
about 4 days before they were given as food. The nudibranch
fed on polyps and hydrocauli of the hydroid. Zooxanthellae
ingested were stored within the liver cells in cerata when the
animals were kept fed continuously. Animals fed on food
showed dark brownish green color in the cerata. When starved
for 2 days, however, the cerata turned to orange indicating
digestion of the zooxanthellae took place. Light and electron
microscopies exmined with starved animals revealed that small
number of the zooxanthellae were remained still in the liver
cells. Adult animal spawned an egg mass every day for more
than 1 month after copulation. The number of eggs in a mass
were over 1200 in fully matured adult. Hatched veligers
provided with well developed digestive and nervous systems
including paired eyes and few radular teeth. They clung to food
hydroid soon after release. After casting off the velar cillia and
larval shell, they metamorphosed to a vermi-form juvenile in the
next day. A pair of cerata developed at first. Sexual maturity
was attained for about 1 month after the hatching.
Unlike the case of symbiosis in nudibranch, Pteraeolidea
lanthina (Kempf,1984) which fed on another zooxanthellated
hydroid, Myrionema cuppressina, this species seems to show a
somewhat temporary association with symbionts ingested.
AN ACOEL FLAT WORM SPECIES RELATED CLOSELY
TO THE SPECIES CONVOLUTRILOBA RETROGEMMA
HENDELBERG AND SSON OCCURS IN OKINAWA
ISLAND,RYUKYU ARCHIPELAGO.
K. Ishikawa! and T. Yamasu? !Coll. of Sci.and Div. of Gen.
Educ., Univ. of Ryukyus, Nishihara,Okinawa.
We collected two acoel species very similar to the species
Convolutriloba retrogemma Henderberg and Akesson (1988) at
two habitats, Cape Zanpa in Okinawa Island and Sesoko Island
in 1991 and 1992. Both species were found in low tidal zone
where colonies of a Hydrozoa Myrionema amboinense Pictet
grew. Onc of them, the larger one, is much more similar to C.
retrogemma than the smaller one, having not only two
transparent spots at eye field and a straight nozzle of seminal
bursa but also coinciding well in size and external morphology.
A tremendous number of adult worms of this species were also
found in an outdoor aquarium in Sesoko Marine Science Center
of University of the Ryukyus in Sesoko Island. However, no
worms have released any progeny upto now.
The smaller species, ranging from 0.62 to 2.43 mm in length,
on the other hand, released so many progenies from the ventral
caudal part that total number of progenies released by an active
individual amounted more than 14 during two week rearing in
the laboratory, at an average rate of 0.8 / day / individual(14
worms were used). Neither transparent spot at eye field nor
sexual organ provided in C. retrogemma and the larger one,
have been found in all specimens collected (270 in total
number) in both habitats so far as examined.
Both species have similar uni-cellular green symbionts with 4
flagella (Prasinophyceae). Motile phase of symbionts separated
from the smaller species seems to be rather shorter in
comparison with those in the larger one or other acoel species
which have also similar flagellated green symbionts.
Whether the larger species might release its progeny or not
should be watched carefully in further observation.
DEVELOPMENT OF THE VERMIFORM EMBRYO OF
DICYEMIDS (MESOZOA) .
H. Furuya, K. Tsuneki and Y. Koshida
Dept. of Biol., Coll. of Gen. Educ., Osaka
Univ., Toyonaka.
The development of vermiform embryos
within the axial cell of Dicyema
acuticephalum was studied microscopically
on the fixed and stained specimens.
An agamate devides equally and its
daughter cells remain in contact with each
other. One of them divides again equally
to produce the prospective axial cell.
This undergoes extremely unequal divisions
at least three times, and smaller cells
degenerate and disappear. From the five
cell stage onward, the division pattern
becomes bilateral type. At the final stage
of embryogenesis, the prospective axial
cell devides equally to produce two
daughter cells, one of which is in-
corporated in the axial cell to form the
agamate. The fully formed embryo is
composed of one axial cell and 18 peri-
pheral cells.
In addition, preliminary survey was
carried out to determine the survival
time of nematogens and infusoriform
larvae of D. japonicum in vitro in the
host octopus urine and also in seawater at
14 C. The nematogens have been alive for
ten days in the urine, but they died
whithin five days in seawater. The
infusoriforms became immobile only within
two days in both the urine and seawater.
1282 Cell Biology and Morphology
ULTRASTRUCTURE OF THE MARGINAL ADHESIVE
ORGANS OF THE FRESHWATER PLANARIANS.
S. Ishii.
Division of Cell Science, Research
Laboratories, Fukushima Medical College,
Fukushima.
A comparative study of the marginal ad-
hesive organs of freshwater planarians
(Turbellaria, Tricladida) was carried out
using 5 species collected in the suburbs
of Fukushima City (Dugesia japonica, Pha-
gocata vivida, Ph. kawakatsui, Polycelis
auriculata and Bdellocephala brunnea).
All the organs examined were of duo-gland
type similar to those reported in marine
forms (Tyler,1976), consisting of long
necks of viscid and releasing glands
whick perforate through the cytoplasm of
special modified epidermal cells, anchor
cells. The secretion granules of the vis-
cid glands were large ovoid dense granu-
les showing multifarious ultrastructural
features of possibly species-specific
varieties. On the contrary, the secretion
granules of the releasing glands were
small and less specific in features.The
anchor cells were of insunk type and pro-
vided with many surface microvilli with
specially developed fibrilar cores which
continued to the cell webs.
The adhesive organ of Bdellocephala
brunnea had specially developed areas
of adhesion, but ultrastructurally they
were basically not different from those
of the other species.
THE BOTRYOIDAL TISSUE IN LEECHES, HIRUDO
MEDICINALIS UNDER 6°C
H.Inamura
Dept. of Biol.,Tokyo Med. Coll., Tokyo
The structure and function of botryoidal
tissue were studied using electron microsc-
opy at temperatures under 6°C,instead of
the previously reported 25°C.Hungry leeches
were fed blood from rabbits once in 2 mont-
hs.After 5 day,the leeches had consumed
four to five times their own weight in
blood with a weight loss of 15 percent,as
compared to a 35 percent weight loss for
leeches at 25°C.Blood in the crop showed
fresh vermilion colors and erythrocyte,in
contrast to blackish colors at 25°C.
In botryoidal cells at 25°C,rER was report-
ed to clearly develop around nuclei,and
in the apical and lateral cytoplasm.
There was lots of microvilli in the lumenal
surface of cells.Cells consisted of three
granule types ;Ll-granules were oil-drople-
ts,L2-g. were ACPase activity,and L3-g.
were like a lysosome.Under 6°C,rER :did not
develop.In the lumenal cytoplasm there
were lots of vesicles with low density,and
microvilli decreased.The apical cell membr-
ane was indented like a tubule with high
density,and was a similar shape at 6°C
and at 25°C.A high numbers of L2 and L3
granules were observed,but Ll-g. were not
observed. After 1 to 4 weeks,cells showed
a similar shape.
Therefore we can conclude that metabolism
in botryoidal cells is affected by tempera-
ture.
THE DEVELOPMENT AND THE REGENERATION OF
THE GENITAL ORGANS IN THE LAND PLANARIAN,
BIPALIUM PENNSYLVANICUM.
Y.Shirasawa and N.Makino. Dept.of Biol.
Tokyo Med. Coll., Tokyo.
The development of the genital organs,
ovary, testis and copulatory apparatus of
the larva and the regeneration of these
organs in the cut pieces of the matured
individual have been examined, morphologi-
cally and histologically, in the land pla-
nNarian, B.pennsylvanicum which reproducts
only sexually. In the three-weeks larva,
we observed several masses which are com-
posed of basophilic cells in the parenchy-
ma between the ventral nerve cord and the
intestine. The anterior small masses make
a pair, the middle larger several ones are
close to the intestine, and the posterior
is the largest, in which the disintegra-
tion of the intestinal wall is most re-
markable, and the stainability of the
basophilic cells is the strongest. In the
next stage, the anterior masses differen-
tiate into ovaries, the middle into testes
and the posterior into copulatory appara-
tus. In the regenerating cut pieces,which
regenerate well without reference of body
levels, the head pieces regenerate testes
and the anlage of the copulatory apparatus
in eight weeks. The pre- and the post pha-
ryngeal pieces regenerate ovaries simi-
larly. A half of the tail pieces regenera-
te heads and pharynges but not the genital
organs.
THE ENDOCRINE CONTROL SYSTEM OF THE
FUNCTIONAL HERMAPHRODITISM OF THE SLUG
(LIMAX MARGINATUS).
N. Seo and N. Makino, Dept. of Biol.,
Tokyo Med. Coll., Tokyo
Until now we have obtained the follow-
ing on the endocrine control of sexual
growth due to neurosecretory cells(NCs) in
the cerebral ganglion(CG) and the suboeso-
phageal ganglion(SG) of Limax marginatus.
The CG and SG at the mature stage were
effective in development of male and fema-
le phases of ovotestes(OT). Female germ
cells (GCs) defferentiated under an unhor-
monal condition. Aldehyde fuchsin positive
NCs were classified Type] — X according
to their size and distribution. TypeVl NCs
appeared during the oviposition stage.
Others appeared in a less mature stage and
increased in number, the number reached a
Maximum in the mature stage.
Endocrine effects on the OT were class-
ified, the provocation of differentiation
of male GCs,the stimulation of progressive
spermatogenesis, the provocation of ovipo-
sition and the acceleration of vitello-
genesis. It can be said that NCS produce
two stimulating hormones of OT, one being
the male differentiation hormone(MDH), the
another being the oviposition hormone(OH).
The functional hermaphroditism is
retained due to autodifferentiation of
female GCs and differentiation of male GCs
by MDH, and by, parallel development of
male and female GCs by increase in MDH and
the hormone of stimulative vitellogenesis.
Cell Biology and Morphology 1283
STRUCTURE OF THE OVARY AND OOGENESIS IN
SCHIZOMUS SAWADAI (ARACHNIDA; SCHIZOMIDA)
K.Miyazaki!, R.Ueshima2 and T.Makioka@.
Dept. of Biol., Keio Univ., Yokohama, and
2Inst. of Biol. Sci., Univ. of Tsukuba,
Ibaraki.
In most orders of the Arachnida, struc-
ture of the ovaries and the modes of oogen-
esis share some unique features (Makioka,
1988). In the Schizomida, however, few
studies have been done on these subjects.
In the present study, we report structure
of the female reproductive system and
oogenesis in a Japanese schizomid, Schizo-
mus Sawadai.
The adult female reproductive system con-
sists of a single sac-like ovary, paired
lateral oviducts, a common oviduct and a
genital pore, all of which are localized in
the abdomen. No oogonia are found in
ovaries of all the examined adult speci-
mens. Very young oocytes are embedded
within the dorsal ovarian epithelium, but
the larger ones protrude outward from the
lateral and ventral ovarian epithelium into
the hemocoel, connected with the ovary by
cellular stalks. A real connection between
the ovarian lumen and the genital pore
through the oviducts indicates that mature
oocytes on the stalks should be ovulated
into the ovarian lumen and sent toward the
genital pore. Structure of the female
reproductive system and mode of the oogene-
sis in S. sawadai are basically identical
with those in many other arachnids, espe-
Cially those in orders of the Pedipalpi.
COMMON FEATURES IN OVARIAN STRUCTURE OF
SOME JAPANESE PENICILLATE DIPLOPODS.
K. Yahata and T. Makioka. Inst. of Biol.
Sci., Univ. of Tsukuba, Tsukuba.
In a European penicillate diplopod,
dagurus, Kubrakiewicz (1989)
described the following structural features
of the adult ovary as the characteristics
peculiar to the penicillates; existence of
a distinct germarium including oogonia and
absence of cellular connections between the
Ovarian epithelium and the oocytes growing
in the ovarian lumen. In adult ovaries of
some Japanese penicillates, we found not
only the germaria, but also the cellular
connections between the ovarian epithelium
and the oocytes.
Eudigraphis nigricans, E. takakuwai,
E. kinutensis, BE. sp., PB. shinoharai, and
PB. sp. showed mostly similar structural
features on adult ovaries despite some
minor differences possibly due to their
sizes. A germarium composed of oogonia,
very young oocytes and interstitial cells
was usually located in the centre of the
ventral ovarian epithelium. Paired germ
areas consisting of young oocytes and young
somatic cells were serially arranged in the
ventral ovarian epithelium, from each of
which areas several elongated somatic cells
rose to be connected with some larger
oocytes floating in the ovarian lumen.
These connecting structures would be common
not only among penicillates, but also to
other diplopods.
POSTEMBRYONIC DEVELOPMENT OF THE OVARY
IN A NOTOSTRACAN, TRIOPS LONGICAUDATUS
(BRANCHIOPODA, CRUSTACEA).
H. Ando and T, Makioka. Inst. of Biol. Sci., Univ. of
Tsukuba, Tsukuba.
Two remarkably different types of ovaries have
been known in the branchiopod crustaceans: one type seen
in the Notostraca and Conchostraca and the other in the
Anostraca and Cladocera. The ovary in the former type
branches into many ovarioles, on the tips of which the
germaria including oogonia are located. An egg-follicle
with a growing oocyte and nurse cells is protruded from
each germarium to the hemocoel. On the other hand, the
ovary of the latter type does not branch. The germarium is
localized at a particular part of the ovary, and the oocytes
grow in the ovarian lumen.
We studied postembryonic development of the
ovary in anotostracan, Triops longicaudatus, to know
how the ovary of the former type, unique among the
Crustacea, is formed and to find origins of some
differences between the types. By the 4th instar, a pair of
ovaries appeared as young germ cell-masses on both sides
of the gut. At about the 11th instar, each ovary became
tubular, having in the ovarian epithelium many germ cell-
masses or germaria with the egg-follicles. This ovary
already showed the outward migration of the growing egg-
follicles as one of the particularities of the former type.
Formation of the ovarioles began later at about the 15th
instar, and spawning eggs at about the 18th.
STRUCTURE OF THE FEMALE REPRODUCTIVE SYSTEM
IN ARGULUS JAPONICUS (CRUSTACEA: BRANCHIURA) .
K.Ikuta and T.Makioka. Inst. of Biol. Sci.,
Univ. of Tsukuba, Tsukuba.
The adult female Argulus japonicus has
a single sac-like ovary lying above the
alimentary canal in the thorax. A long germ
zone or germarium including many oogonia
runs in the dorsal ovarian epithelium along
the median line. A pair of wide and flat-—
tened oviducts start from the antero-dorsal
end of the ovary and run along both sides
of the ovary. Either of oviducts is con-
nected with a genital pore at the end of
the last thoracic segment, and the other
ends blindly. Growing oocytes are found,
not in the ovarian lumen, but on the outer
surface of the repeatedly folded ovarian
epithelium, protruded into a narrow hemo-
coelic space surrounded by the oviducts and
the alimentary canal. These oocytes are
tightly covered with the extended basement
membrane of the ovarian epithelium. Neither
follicle cells nor nurse cells are found
around the oocytes. Mature eggs are ovu-
lated into the ovarian lumen, transported
into and stored in the oviduct connected
with the genital pore. A pair of sperma-
thecae are located in the abdomen, opened
just outside the genital pore.
Some basic features on structure of
the ovary and oogenesis in A. 3 j
seem similar to those in many chelicerates
and pentastomids rather than those in many
other crustaceans.
1284 Cell Biology and Morphology
STUDIES ON THE ENDOPARASITES OF ANURA
5.0N THE NEMATODA;RHABDIAS NIPPONICA IN LUNG Of RANA
Y.Sasaki and N.Makino. Dept. of Biology.,Tokyo Med.
Coll., Tokyo.
The nematoda, Rhabdias nipponica is known to show
the hetelogony.This worm of parasitic stage are fe-
nale. Eggs are deposited by the female,hatch out,
and the hatched larvae develop and grow up to free
living males and females.We tried to culture of this
hatched larvae. Under the environment of water as
a basis, larvae were in high spirit for two or three
days but after five or ten days,their half died.
Only a few larvae lived fourteen days. Many larvae
didn’t grow and their body length was about 500un.
Sometimes big larvae were observed, their body length
was about 700 um and they were two times as body
width as many larvae. It seems that big larvae grow.
And then hatched larvae were cultured under various
following solution ; frog’s ringer, Earl’s balanced
salt solution and medium 199 . FBS was added 5% or
10% to culture media to achieve a final concentra-
tion. And antibiotics were that a mixture of peni-
cillin and streptomycin was added to culture media
to achieve a final concentration of 100 or 50 units
penicillin and 0.1 or 0.05 mg streptomycin per ml
medium. In the each culture media,survival of larvae
were the same number of days with water as a basis.
It is interesting that a few adult worms were sur-
vived about one month in some culture media. But
eggs of this females showed a poor rate of hatching.
CYTOCHEMISTRY AND ULTRASTRUCTURE OF "MYCE-
TOMES" OF THRIPS (INSECTA, THYSANOPTERA }
Tsutsumi, M. Matsuzaki~ and K. Haga
Inst.of Biol.Sci.Univ.Tsukuba, Tsukuba.
Lab.of Biol.,Fac.of Educ. ,Univ.Fukushima,
Fukushima.
Idolothripine thrips, Bactrothrips
brevitubus possesses the structures named
mycetomes" in the oocyte or egg, which
have been accepted as the intracellular
symbiont from the morphological appearance.
It was, however, revealed through our study
that the "mycetome" of thrips is different
from the structures identified as intra-
cellular symbiont in the other insects in
some features, such as stainability to
basophilic dyes [hematoxylin, Schiff's
reagent (Feulgen's reaction) etc.]. Here,
we examine the "mycetome" in detail cyto-
chemically and ultrastructurally, aiming at
the characterization of it.
From the present study, the features
of "mycetome" are summed up as follows:
1) The "mycetome" is composed of numerous
small granules filled with electron-
dense material and larger ones with
myelin figures inside.
2) The "mycetome" shows positive stain-
ability to the dyes of which specific
uptake into lysosome is demonstrated
(neutral red, acridine orange etc.).
3) High activity of acid phosphatase is
detected in the "mycetome" (metal
precipitation or azo coupling method).
These results may suggest that the
thysanopteran "mycetome" should be not
symbiont but an aggregation of lysosomes.
CHARACTERISTICS OF THE PHOTORECEPTIVE CELL
CANDIDATE IN THE BUTTERFLY GENITALIA
Y. Miyako, K. Arikawa, and E. Eguchi. Dept of
Biol, Yokohama City Univ, Yokohama
Two pairs of extraocular photoreceptive
sites (Pls and P2s) were identified in the
butterfly genitalia. Previously we reported that
a distinguishable ovoid structure (ca.30x40pm)
containing a cell body of a sensory neuron
(photoreceptor candidate;PRC) exists in both
sexes' Pls of Papilio xuthus?. At the distal Part
of the sensory neuron(PRC) there are several
distal processes(DP) and a plenty of tubular
membranes (TM) protrude from the tip of the DPs.
They form an entangled thread ball-like structure.
In the present study to examine the effect
of light to the ultrastructure of PRC, the
genitalia of the intact female Papilio xutus were
illuminated by white light of ca.20000lux for 4
hours, then Pl was examined by electron
microscopy. As a result in the distal part of the
PRC tightly packed TM disappeared and a number of
vesicles and membranous fragments became abundant
in several specimens.
In addition the nerve which innervates to Pl
of each sex, mN6-2 for male and £N6-3 for female
was identified. While recording the photoresponse
from the nerve of mN6-2 or from £N6-3 with
isolated Pls, nervous tissue in Pis were
Surgically isolated from the cuticular surface and
exess tissues were removed. Resulting piece of
tissue that still respond to the light flash with
the sustained train of spikes was fixed, embedded
in Epon and examined by electron microscopy. As a
result the preparation contained the PRC.
The results suggest that the PRC is the
genital photoreceptor.
1.Arikawa et al. Nature, 288,700-702, 1980
2.Miyako et al. Zool mag, 7,1028, 1990
MORPHOLOGICAL CHANGES AND MITOTIC
ACTIVITY OF DUODENAL MUCOSA IN FASTED
XENOPUS
K.Fujikura, T. Sakai, and Z. Itoh. Inst. of Endocrinol. Gunma
Univ., Maebashi.
The epithelial cells of the intestine continuously proliferate,
but they are constantly maintained in its size. The mechanism
involved here is not well known, but stimuli brought by food
are considered to be one of the important factors. In the
present study, we used Xenopus as an experimental animal
because it remains healthy without taking food for long
period. We examined morphological changes and the mitotic
activity in the duodenal epithelial cells of Xenopus after they
were fasted for 14, 28, 42, 77, 109 and 151 days, and observed
the effect of re-feeding. It was found that the mean height of
villi in the duodenum measured by means of a microscopic
image analyzer gradually decreased with time. On the 28th
day of fasting the mean height had significantly decreased and
was only 1/2 of the control by day 151. But the mean number
of villi in the circumference of the duodenum did not change
at any days of fasting. On the other hand, the mean frequency
of mitosis detected by immunohistochemical staining with
bromo deoxyuridine labeling rapidly decreased after fasting
for 14 days, and remained at 20-30% of the control all
experiment days thereafter. However, re-feeding caused a
rapid increase in the frequency of mitosis, which reached 2.5
times after 5 days of re-feeding. These findings suggest that
the growth of epithelial cells and frequency of mitosis of the
duodenal mucosa are significantly affected by food intake.
Cell Biology and Morphology 1285
HISTOCHEMICAL STUDY ON THE DERMAL CHROMATO-
PHORE UNIT FORMATION IN THE FROG, HYLA
ARBOREA. 2 1
M.Yasutomi and Y.Yokota . Biol. Lab.,
Aichi Med. College, Aichi and “Biol. Lab.,
Aichi Pref. Univ., Nagoya.
In the frogs of the genus Hyla, the der-
mal chromatophore unit (DCU) was formed
during metamorphosis and the animals have
ability for rapid color change (physio-
logical color change). When metamorphosis
began, the fibroblasts, which were seen
under the basal lamina (BL) in the tadpole,
invaded into BL and open space (stratum
spongiosum;SS) was formed. Chromatophores
such as melanophore, xanthophore and irido-
phore, were also present under BL in the
tadpole and migrated through BL at metamor-
phic stage and reached to SS. Then, DCU
was formed. The sections of the back skins
were incubated with peroxidase-labelled
lectins (ConA, WGA, RCA and PNA). RCA and
PNA bindings were observed in SS. Immuno-
electron microscopic study using anti-
laminin IgG showed the existance of
laminin in BL. The direction of the neu-
ral crest cells migration was determined
by galactose bearing proteoglycans and
laminin (Milos et al.,1986; Bronner-Fraser,
1987; Martin-Green & Erickson,1987). These
facts suggest that the migration of the
chromatophores and the DCU formation
involve the interactions between chromato-
phore surface components and galactose
bearing proteoglycans and laminin.
THE EFFECTS OF TPA AND CHOLERA TOXIN ON FROG
MELANOPHORE PROLIFERATION IN CULTURE.
S. Takeuchi, H. Suzuki, M. Yabuuchi and
Y. Kobayashi. Dept. of Biol., Fac. of Sci.,
Okayama Univ., Okayama.
The present study was undertaken to compare
and correlate the effects of 12-O-tetradecanoyl-
phorbol acetate (TPA) and cholera toxin(CT) on
frog me lanophore proliferation. Dermal
melanophores were isolated from the dermis of a
tree frog (Hyla arborea) and cultured in diluted
Leibovitz-L15 medium supplemented with 20% FBS
and 16nM TPA and/or 2nM CT. TPA and CT
concomitantly induced the cell proliferation at
a maXimal rate and the cells finally piled up as
previously shown. TPA alone in the absence of CT
did not result in optimal proliferation and the
contact inhibition was observed. These results
suggest that mitogenic effect of TPA on frog
melanophore was greatly enhanced by the addition
of CT. Under our experimental conditions,
proliferating cells shared a common
morphological features which included dispersion
of melanosomes in the cell. This response
resembled that induced by CT and hence appears
to be cAMP mediated, however, CT did not induce
melanophore proliferation. Further analysis are
required to determine the exact role of protein
kinase C and protein kinase A in regulating
proliferation of frog melanophores.
RETINOIC ACID DIRECTLY AFFECTS PROLIFERA-
TION AND DIFFERENTIATION OF FETAL RAT FORE-
STOMACH EPITHELIAL CELLS IN PRIMARY CULTURE
H. Fukamachi. Zool. Inst., Fac. of Sci.,
Univ. of Tokyo, Tokyo.
We have previously reported that fetal
rat forestomach epithelial cells prolifera-
te in primary culture. We have also shown
that retinoic acid (RA) inhibits forestom-
ach epithelial keratinization in organ
culture. Here we examined whether RA
directly affects forestomach epithelial
cells in primary culture.
Forestomach epithelial cells were
cultured as previously reported. MTT
assay showed that their proliferation was
suppressed when more than 2 mcg/ml of RA
were added to the culture medium. Thus,
the effect of RA on the epithelial differ-
entiation was examined by adding RA on day
4 in culture when the cells have prolifer-
ated and formed cell sheets. In control
cultures, the cells exhibited keratiniza-—
tion on days 6 to 7. Their keratinization
was suppressed when 20 mcg/ml of RA were
added to the culture medium composed of F12
and growth factors, but such effect was not
observed when MDCK152 was used instead of
Fl2. EXpression of AE3 cytokeratin antigen
was also suppressed in RA-added cultures.
We thus conclude that RA directly affects
both proliferation and differentiation of
fetal rat forestomach epithelial cells, but
that many other factors are involved in the
control of the epithelial keratinization.
PRIMARY CELL CULTURE OF APHID DERIVED CELLS
H. Harada and H. Ishikawa. Zool. Inst.,
Fac. Sci., Univ. of Tokyo, Tokyo.
Aphids, as pests of various crops and
the most important group of plant virus
vectors, have attracted investigators’
interest for a long time. In spite of every
effort made to establish a cell line of
aphid, there is no succesful report, yet.
Aphids have bacterial endosymbionts
which are harbored by bacteriocytes in the
fat body. Endosymbionts are indispensable
to normal growth of aphids, and do not have
free living phase any longer.
In an attempt to study this host-
endosymbiont interaction in the aphid
symbiotic system, we tried to establish the
primary cell culture system of this insect.
We were successful in establishing a
culture system free from gut microbes,
which enabled us to culture the aphid cell
and its endosymbiont without antibiotics.
Among seven insect media tested, the TC-100
medium exhibited the best survival of aphid
cells. Five percent FBS included in the
TC-100 insect medium activated the protein
synthesis of aphid cells, and promoted
cell attachment. On the contrary, the
isolated endosymbionts in the medium with
FBS were broken down after aggregated to
each other within several days.
1286 Cell Biology and Morphology
CELL CULTURE OF KERATINOCYTES FROM CHICK
EMBRYONIC TARSOMETATARSAL SKIN AND THE
DECREASE OF pH IN THE CONDITIONED MEDIUM.
K. Mikami-Takei, H. Endo and A. Obinata.
Dept. Physiol. Chen., Fac. Pharmaceu.
Sci., Teikyo Univ., Kanagawa.
It was impossible in MEM+FCS medium to
culture chick embryonic keratinocytes but
in BGJb medium, which has been used in
organ culture of chick embryonic skin with
supplementary chicken serum for several
weeks though they keratinized autonomously
and their rates of increase were rela-
tively low. When cultured keratinocytes
were mixed with skin fibroblasts, they
keratinized more quickly but with fibro-
blasts that ceased proliferation by hard
X-ray-irradiation or by treatment with
mitomycin C, keratinization was reduced.
These data suggest that the chick kerati-
nocytes culture requires a milieu of which
the cells come from and fibroblasts sup-
port keratinocytes culture effectively.
It is also observed that marked pH de-
crease in the conditioned media of kerati-
nocytes with fibroblasts. Any increase of
organic acid was not detected by HPLC
analysis. Amino acid analysis revealed
the decrease of glutamine in the condi-
tioned media. The reason why mix culture
decreased pH of media is unknown, but this
Phenomenon implies some interaction be-
tween keratinocytes and fibroblasts.
FCS INDUCED CELL DEATH IN VITRO
Takeshi Kurita and Hideo Namiki. Dept. of Biol.,
Sch. of Educ., Waseda Univ., Tokyo.
Serum is commonly added to defined basal
media as a source of nutrients and macro-
moleculars essential for cell growth. Fetal
calf serum (FCS) has been believed as the best
one for cultures and used most commonly. It is
well known that FCS often displays growth
inhibition and cytotoxic-effects. The mechanism
of the FCS cytotoxity is however not clear and
not investigated well.
We found high content of FCS in the basal
medium induced cell death in several types of
cultured cells. In order to estimate’ the
morecular-weight of the toxic factor(s), FCS was
separated into two fractions by ultrafiltration
(YM2 M.W.1,000cut, Amicon). The macromolecular
fraction was supplemented inorganic salts and
nutritents and its osmotic-pressure was adjusted
with MEM. Thus reconstituted medium (UF-FCS)
didn’t induce cell death. On the other hands,
high consentration of the FCS filtrate («1,000)
induced cell-death. These data suggest FCS
containes low-molecular-weight(<1,000) factor(s)
which causes cell growth inhibition and cell
death. As far as we tested, all type of cells in
culture necrosed in FCS and well grew in UF-FCS.
DISASSEMBLY OF F-ACTIN FILAMENTS IN HUMAN
ENDOTHELIAL CELLS ON TYPE V COLLAGEN
K. Yamamoto+, M. Yamamoto+-~, and Ths
Noumura~
+Dept. Cell Biol., Tokyo Metropol. Inst.
Gerontol., Tokyo, “Dept. Regul. Biol.,
Fac. Sci., Saitama Univ., Urawa.
Human endothelial cells cultured on
type V collagen attached temporarily
to the substrate and formed F-actin
filaments. However, the cells failed to
proliferate and gradually detached from
the substrate. After 24 h, the cells on
type V collagen lacked discernible F-
actin filaments and exhibited dots in
small aggregates of F-actin. In addition,
the cells expressed little or no proteins
as focal adhesions, including vinculin
and f, integrin. In contrast, the cells
on fibronectin and type I collagen
developed complete F-actin filaments,
exhibited sufficient vinculin and B
integrin, and grew logarithmically from 4
days. On the other hand, human smooth
muscle cells formed complete F-actin
filaments, revealed typical focal
adhesions, and started to grow rapidly
after 24 on type V collagen as well as on
fibronectin and type I collagen. Thus,
the disassembly of F-actin filaments in
focal adhesions was observed as a
specific phenomenon in human endothelial
cells cultured on type V collagen and may
result in the detachment of the cells
from type V collagen.
AUTOCRINE REGULATION OF THE MIGRATION OF
HUMAN FETAL SKIN FIBROBLASTS.
H. Kondo and Y. Yonezawa, Dept. of Exp.
Biol., Tokyo Metropol. Inst. of Gerontol.
Tokyo.
It was recently reporte that growth
factors in addition to extracellular
matrix (ECM) were extremely important to
cell migration. We found out using Stenn's
method, which was devised as a model sys-
tem of wound healing, that adult-donor
skin fibroblasts migrated more slowly in
serum-depleted medium than in serum-
supplemented medium. On the other hand,
human fetal skin fibroblasts migrated in
serum-free medium as well as in serum-
supplemented medium. In other words, human
fetal skin fibroblasts migrate autonomous-
ly.’To determine what factors regulate the
migration of human fetal skin fibroblasts,
we investigated whether monensin, an in-
hibitor of ECM secretion and suramin, a
competitor of growth factors at the
receptor level suppressed fibroblast
migration. The following results were
obtained. Monensin inhibited the migration
of human fetal skin fibroblasts. And,
addition of type I collagen abolished the
inhibition by monensin. Suramin also
suppressed fibroblast migration. This
inhibitory effect was partially recovered
by addition of PDGF or bFGF. These results
indicated that the migration of human
fetal skin fibroblasts was regulated by
growth factors and ECM which were supplied
by an autocrine manner.
Cell Biology and Morphology 1287
EFFECTS OF SIMIAN VIRUS 40 LARGE T-
ANTIGEN GENE ON CELL IMMORTALIZATION.
N. Yanai, R. Okuyama, and M. Obinata.
Dep. of Cell Biol., Res. Inst. for
Tuberculosis and Cancer, Tohoku Univ.,
Sendai.
We established several cell lines
from transgenic mice harboring tempera-
ture-sensitive Simian Virus 40 large T-
antigen gene. Those cell lines exhibit-
ed continuous proliferation under the
permissive temperature with their dif-
ferentiation phenotypes. A liver cell
line (TLR2) had potencies to produce
albumin and was induced P450IAl1 by 3-
methylcholanthrene. A tubule cell line
(TKC2) were induced intracellular cAMP
by arginine vasopressin. At the nonper-
missive temperature, the degradation of
T-antigen was observed, and cells could
not proliferate and successively dead of
apoptosis. According to previous works
revealed that cell immortalization is
caused by mutations of recessive immor-
talizing genes. Presented result may
lead a hypothesis that SV40 T antigen
cause not only cell proliferation but
also blocking of cell death, then pre-
vent a senescent stage before an immor-
talization.
SUPPRESSION OF UV-INDUCED CELL TRANSFORMA-
TION BY A CONTACT-INHIBITORY FACTOR IN
MOUSE C3H10T1/2 CELLS.
T.Yamaguchi?., and K.Nakasone THace Gent
Educ. and *Fac. Sci., Ehime University.,
Matsuyama. (“Present address: Japan Inter-
national Cooperation Agency, Tokyo)
2x
,
A membrane fraction of C3H10T1/2 (cl.8)
cells prepared by homogenization and
successive centrifugation was effective to
inhibit the proliferation of a sparsely
seeded growing culture of the same cell
line which was sensitive to the contact
inhibition of growth. The cells in conflu-
ency were irradiated with UV, plated
sparsely and treated with the membrane
fraction during the transformational
damage-fixing period (1 week) after the
irradiation. The culture was continued
with a weekly medium renewal for 6-8 weeks
and transformed foci were detected. The
transformation frequency was expressed as
the ratio of the transformed foci to the
number of surviving colonies using a
formula of Poisson distribution.
The transformation frequency was sig-
nificantly decreased in a dose-dependent
Manner by the membrane fraction added,
suggesting the suppression of carcinogene-
sis by the inhibition of cell prolifera-
tion during the damage-fixing period after
irradiation: A similar condition might be
assumed to exist within the body where a
damaged cell was surrounded by other cells
of the same tissue.
IN VITRO MODEL FOR THE PERIPHERAL AUTONOMIC NERVOUS
SYSTEM : DIFFERENTIATION OF NEURITES
T.MARUYAMA and Y.ENDO
Dept.of App].Biol., Kyoto Institute of Technology,
Matsugasaki, Sakyo, Kyoto
In order to construct a model in vitro for the
peripheral autonomic nervous system, we have tried to
co-culture a pheochromocytoma cell line (PC-12) as a
Neuronal element, and a smooth muscle cell line (SM-3)
and insulinoma cel] line (In-R1-G9) as target cells.*
PC-12 cells elongated several thin processes in this
system, which appeared to be somewhat different in
shape each other. It suggested the possibility of
differentiation of them into axons and/or dendrites.
An immunocytochemical approach using monoclonal
antibodies agaist microtubule-assocated proteins
(MAP1,2) showed that some neurites have no
immunoreactivity of MAP2, whereas most of them have
MAP1-immunoreaction. Occurrence of axon-like neurites
without. MAP2 immunoreactivity was seemed to be related
with their contact or adhesion to the other cells.
Alternatively, length of neurites was also seemed to be
related with the differentiation of neurites.
Cells were also cultured by embedding in the typel
collagen gel, just like in vivo conditions. Although
the collagen gel caused a conspicuous change of cell
shape, we could not find the difference of MAP
distribution.
xy. ENDO, T. MARUYAMA, Y.SASAKI (1991) Biomed Res 12:211-214
DEVELOPMENT OF NERVOUS SYSTEM IN THE
TELEOST FISH, ORYZIAS LATIPES WITH SPECIAL
REFERENCE TO RADIATION EFFECTS.
Y. Hyodo-Taguchi and Y.Ishikawa. Div. of
Biol. Natl. Inst. of Radiol. Sci., Chiba.
The process of normal development of
nervous system in the brain region of the
medaka, Oryzias latipes and its radiation
effects were preliminary studied. Embryos
and fry of inbred strain HO4C and albino
medaka were used. To examine development
of nervous system, we made whole-mount
specimens and the nerve fivers were
stained immunohistochemically by using
antineurofilament protein(NFP) antibodies.
Divisions of the brain into the fore-,
mid-, and hindbrain became well-defined at
St.22. Mauthner cells(MC), nucleus of
fasciculus longitudinalis medialis (FLM)
and FLM were identified by NFP staining at
St.24. At the same stage, 4-5 pairs of
the neurons of reticular formation and
reticulospinal nerve fibers (RF) were
observed in the hindbrain region. Until
hatching stage, all cranial nerves were
formed. If doses of 2-6 Gy of radiation
(X-rays, heavy ion and Y-rays) were given
to embryos in early developmental stages,
disorder of bilaterally symmetrial
structures of nervous system was often
occurred in the embryos with morphological
anomaly.
1288 Cell Biology and Morphology
RADIOBIOLOGY OF THE GUPPY, LEBISTES
RETICULARIS (PETERS), THE RADIATION DEATH
INDUCED BY WHOLE-BODY IRRADIATIONS WITH
X-RYAS AND CARBON ION PARTICLES.
H.Ohara, Y.Kohda, T.Kanai, Dept. Gen. Ed., Okayama Univ.,
Okayama, and Div. Accel. Res., Natl.Inst.Radiol.Sci., Chiba.
The young guppies, being aged about 2-3 months after birth,
were received whole body irradiations with graded dosis of 200
kVp of X-rays and 135 MeV/n of carbon ion beams produced by
the RRC at RIKEN(Inst. Chem. Phys.). The fishes were
observed for the post-irradiation occurrence of radiation death
over 6 months of period. The possible reasons of radiation death
were also explored by histological examinations in various
tissues and organs. The main results were as the followings.
More than 95 % of fishes died after irradiation with 15 Gy of
heavy ions, while more than 60 % of fishes were survived by 25
Gy of X-rays. The most sensitive tissues/organs were identified
as hematopoetic kidney tissues and those of epithelials in skin,
gill, intestine, and oral cavity. Testis, liver, and glomerulus in
kidney were also identified as being sensitive. Remarkable loss
of blood cells was found in the head kidney , but loss of villi was
not so distinct even with higher dosis of heavy ion irradiation.
The damage of epithelial tissues in skin and oral parts was much
more severe than those of intestine. Accordingly, some fishes
died apparently from the starvation of foods due to the damage
of oral cavity epithelials. This can be identified as "oral death" ,
which has already defined as the most important reason of
radiation death in mammals to be induced with almost same level
of radiation dosis. RBE for cabons was evaluated as some of 2.0
-2.2 in occurrence of radiation death at 20 C.
IMP-DISTRIBUTION ON PLASMA MEMBRANE OF FISH
EGGS BEFORE AND AFTER CORTICAL ALVEOLUS
BREAKDOWN
T. Ohta! and E. Kinoshita?, Dept. of Life Sci.,
Aichi Univ. of Educ., Kariya, *Fac. of Integr.
Arts and Sci., Hiroshima Univ., Hiroshima.
Cortical alveolus breakdown (CABD) of the
rose bitterling (Rhodeus oce/latus ocellatus)
eggs occurred in fresh water and continued
for about 20 min. Insemination in fresh water
induced polyspermic fertilization in all
dechorionated and non-activated eggs. At 15,
30 and 60 min after activation treatment (AT,
immersing into fresh water). dechorionated
eggs were inseminated. The results showed that
polyspermic fertilization rates decrease with
the time after AT. Membranes of CA were
added to egg plasma membrane (EPM) with CABD.
We focussed on the change in IMP-distribution
on the plasma membrane to clarify for the
decrease in the fertilization rates.
In non-activated eggs, few IMPs were
distributed on PF surface of EPM and many IMPs
existed on the PF surface of CA. The diameter
of IMPs was about 13 nm. In eggs 15 min after
AT, EPMs were dotted with aggregates of slight
increased IMPs. In the eggs 30 and 60 min after
AT, IMP-distribution on EPM was similar to that
of non-activated eggs, in spite of addition of
CA IMPs. No changes in IMP-distribution which
seem to result in decreased fertilization rates
were observed.
CTENII FORMATION IN REGENERATING
CTENOID SCALES IN THE JAPANESE
FLOUNDER, PARALICHTHYS OLIVACEUS.
Ss. Kikuchi? ,H. Nakamura* and A.
Shimozawa~ {Kominato Lab.,Fac. of
Sci., Chiba Univ., Chiba, #Dept. of
Anat., Dokkyo Univ. Sehr of Med.,
Tochigi.
Squamation process of regenerating
ctenoid scales of the Japanese
flounder Paralichthys olivaceus were
studied histologically with special
reference to ctenii formation. Scales
were plucked off from ocular sides of
fish and after several intervals,
regenerating scales were observed by
light microscopy (LM) and scanning
electron microscopy (SEM). For SEM,
aldehyde-fixed specimens were
immersed in a solution of diluted
sodium hypochlorite to remove soft
tissues which covered ossified
scales. On 5th day of regeneration,
minute calcium deposition was stained
with alizalin red. By SEM, 2 to 6
circuli and small ctenii were already
found on the edge of the scale
plates. Subsequent ctenii formation
was observed between pre-formed
ctenii and this formation patern
“might make cone-shaped ctenii of
ctenoid scales.
SYNAPTOLOGY OF SPINAL MOTONEURONS IN BONY
FISH : COMPARISON BETWEEN LARGE AND SMALL
NEURONS.
A. Matsumura. Dept. of Biol., Natl. Def.
Med. Coll., Tokorozawa.
The author previously presented the
ultrastructure of large motoneurons dis-
tributed from the lateral side of the
central canal to the anterior horn of the
spinal cord in goldfish, Carassius auratus.
In the present study, the neurons distrib-
uted in the same area and the cross-
sectional area of the cell body up to
440 pm? were determined as small motoneurons,
and the fine structure waS examined by
comparing it to large motoneurons using a
transmission electron microscope. In the
densities of the cell organelles, the S/F
ratio ( large cells, 1.49; small cells,
We 3) was not largely distinguished
between the neurons, but the covering
ratio of the cell body by terminal boutons
was 46.1% in the large neurons and 19.8%
in the small ones and the ratio of the
small neuron was less than half of the
large ones. Even considering the size of
the cell body, there were large quanti-
tative differences in the total synaptic
inputs between the large and small neurons.
This study was co-worked, with Dr. K. Saito.
Cell Biology and Morphology 1289
A HISTOLOGICAL STUDY ON THE NERVE INNERVA -
TION IN THE ORAL CAVITY OF CLEMMYS JAPONI -
CA.
H. Ishihara. Biolog. Lab., Fac. of Sci &
Engineer., Aoyama {rakuin Univ., Tokyo.
The morphology of nerve innervation and
nerve endings in the oral cavity of Clemm-
YS japonica was investigated according to
the silver impregnation method devised by
the present author. Oral cavity walls
were constituted of tunica muscularis,
tela submucosa and epithelium. It was
found that the nerve plexus in oral cavity
consisted of both autonomic and central
nerve fibers showing wavy course. Emerged
from fundus plexus, the nerve bundles were
observed to enter tunica muscularis and to
form plexus myentricus. The nerve bundles
originated from plexus myentricus, after
repeated ramifications and anastomosis came
to form fine net-work, and then constitute
a fine terminal reticulum were seen.
Further in this net-work, many ganglion
cells were detected here and there. Cap-
sulated Slomerular corpuscles were found to
occur in the smoothe muscle layer. In the
tela .submucosa, the nerve fibers branched
from the secondary net-work, further again
branched out the fibers which terminated in
this layer were detected. Fine neurofi-
brils running freely in all directions were
very often found. In addition, fine nerve
fibers were observed to enter the epithe-
lium and to end there.
A COMPARATIVE STUDY ON THE MITRAL AND
GRANULE CELLS OF THE OLFACTORY BULB.
N. Iwahori, Dept. of Anat., Fac. of Med.,
Nagasaki Univ., Nagasaki.
The intrinsic organization of the
olfactory bulb (OB) was studied in the
mouse and arctic lamprey with special
reference to the morphology of the mitral
(MC) and granule cells (GC).
The OB in the mouse showed clear
laminar organization. The mouse MC had
primary and secondary dendrites: the
former traveled superficially to be
distributed in the olfactory glomeruli,
while the latter extended in an arched
manner forming the external plexiform
layer. The processes of the GC traveled
superficially to terminate mainly in the
external plexiform layer. The lamination
of the OB in the arctic lamprey was much
more obscure than that of the mouse OB,
and the external plexiform layer could
not be identified. The MC of the arctic
lamprey had several primary dendrites but
secondary dendrites were not observed. The
GC in the arctic lamprey had several long
superficial processes which terminated in
the olfactory glomeruli forming tufted
endings.
Thus, the morphology of the MC is
closely related to the laminar patterns
of the OB, especially to the external
plexiform layer, and also to the terminal
areas of the GC processes.
REARING ENVIRONMENT EFFECTS ON SYNAPSES IN
VOMERONASAL SYSTEM
M. Ichikawa: Dept. of Anatomy and Embryology, Tokyo
Metropolitan Inst. for Neuroscience, Tokyo
The effects of differential rearing on synaptic
morphology were examined in the granule cell layer of
accessory olfactory bulb (AOB) of adult rats. Forty-day old
male rats were housed in one of three ways: individually
(isolated condition-IC); with 4 males per cage (unisexual
condition-UC); or with 2 males and 2 females per cage
(social condition-SC). After 2 months, the animals were
prepared for electron microscopy. Two type of synapses
were Classified; (1) perforated (P) synapses, which are
characterized by discontinuities in the postsynaptic
thickening, and (2) nonperforated (NP) synapses. The
length of synaptic contact zone (SCZ), and area (A) and
length of perimeter (LP) of presynaptic and postsynaptic
terminals were measured in each synapse. In the P
synapses, the length of SCZ were significantly greater in
the UC and the SC than in the IC. In the presynaptic
terminals, the A was greater in the UC and the SC than in
the IC, and the LP was longer in the UC than in the IC
while, in the postsynaptic terminals, there was no
difference in the A and the LP among three groups. In the
NP synapses, there was no statistically significant
difference in both the A and the LP of presynaptic and
postsynaptic terminals among three conditioned groups
while the length of SCZ was larger in the SC than in the
IC. These results demonstrated that the exposure to
different rearing conditions, in which the pheromonal
environment can be substantially different, can induce
striking morphological changes in the presynaptic terminal
and the SCZ of P synapses in the AOB of adult rats.
DIFFERENT EFFECTS OF ESTROGEN ON THE
INTRACELLULAR LAMININ IN THE ANTERIOR
PITUITARY OF FEMALE AND MALE RATS
T. Kikuta and H. Namiki
Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo
Through immunocytochemistry we (1988) have
demonstrated the quantitative sexual difference of
laminin positive LH cells in the rat anterior pituitary at
puberty. In the case of the female, the number of laminin
positive cells decreased at the age of 40 days, nevertheless,
that of male rats increased at the same age. And we (1991)
reported the instant decrease of laminin positive cells of
prepubertal female (non-OVX) rats provided estrogen.
In the present study, we examined changes in number
of the positive cells of 30 and 60 day-old male and 25 day-
old ovariectomized female rats given steroid hormones (30
days male: estradiol-17f or testosterone; 60 days male and
25days female: estradiol-17$).
As for 30 days male, laminin positive cells increased 1
to 5 day after either the treatment of estradiol or
testosterone, and arrived at maximal level at day 5, as well
as intact 60 days, and then slightly decreased at day 7. But
for 60 days male, estradiol did not seem to have any effects.
In the ovariectomized female, behavior of the positive cells
is similar to the non-OVX, although, the decrease slightly
delayed.
We have thus concluded that the sexual difference of
laminin positive cells, that is, decrease in number of
positive cells in female rats and increase in male rats, are
due to the difference of the pituitary responsiveness to sex
steroids between male and female.
1290 Cell Biology and Morphology
G-S CONNECTION (INTIMATE APPOSITION OF THE
GLOMUS AND SMOOTH MUSCLE CELLS) IN THE
AMPHIBIAN CAROTID LABYRINTH
T. Kusakabe. Dept. of Anatomy, Yokohama
City Univ. School of Med., Yokohama
Intimate apposition of the glomus and
smooth muscle cells (g-S connection) was
found in almost all glomus cells of the
carotid labyrinths in juvenile ullfrogs,
Rana catesbeiana, 1 month after metamor-
phosis. There were three types of g-s con-
nection: between 1) the tips of many
slender processes (0.1-0.2um in width)
without dense-cored vesicles of glomus
cells and smooth muscle cells, 2) between
relatively thick processes (1.0-1.5um in
width) of glomus cells with dense-cored
vesicles and some cell organelles and
smooth muscle cells, and 3) between some
tongue-like projections of smooth muscle
cells and the flat surface of the glomus
cell. In some cases, a single glomus cell
formed g-s connections with two or three
smooth muscle cells. Exocytosis was often
observed at the g-s connection. Enclosed
by a supporting cell, afferent and efferent
synapses with typical membrane thickenings
were found on the glomus cells with g-s
connections. Reciprocal synapses were also
observed. On the basis of these findings,
the second and third types of g-s
connection are presumed to participate in
the vascular regulation of the carotid
labyrinth.
ASSESSMENT OF THE BILATERAL SYMMETRY OF
THE MYELINATED NERVE FIBER NUMBERS IN THE
MOUSE RAMUS STAPEDIUS, R.JUGULOHYOIDEUS
AND R.STYLOHYOIDEUS.
M.Aikawa, E.Furuta and A.Shimozawa
Dept. of Anat., Dokkyo Univ. Sch. of Med.,
Tochigi.
Myelinated nerve fiber count was
performed on the mouse r.stapedius (RSd),
r.jugulohyoideus (RJh) and r.stylohyoideus
(RSh) of n.facialis from both sides of the
individual to examine bilateral symmetry.
The mean fiber number in the right RSd was
75.0+16.9, and in the left RSd it was 77.1
+21.9 (mean+s.d., n=8). The mean fiber
number in the right RJh was 14.0+2.6, and
in the left RJh it was 13.4+2.4 (n=12).
The mean fiber number in the right RSh was
14.9+2.9, and in the left RSh it was 17.0
+3.4 (n=9). The differences in the fiber
number between the right and left nerves
in the same individual were 9.9+4.2 (range
6-18), 1.9+1.4 (range 0-4), and 4.1+3.1
(range 0-10), respectively, for the RSd,
RJh and RSh. The intra-animal relation
between the fiber numbers from the right
and left nerves was close in the RSd and
RJh, but was not close in the RSh.
The correlation coefficient was 0.88 for
the RSd and 0.58 for the RdJh.
These data show that the myelinated
nerve fiber numbers in the right and left
of the same individual are approximately
equal in the RSd and RJh, but are unequal
in the RSh.
A DISTRIBUTION AND LOCALIZATION OF SEROTONIN IMMUNOREACTIVE
CELLS IN THE TRACHEAL EPITHELIUM OF HYNOBIUS TOKYOENSIS.
Y. Kikuchi. T.Gomi, A.Kimura and K. Kishi.
Dept. of Anat., Sch. of Med., Toho Univ.
Tokyo.
The trachea of Hynobius tokyoensis
were studied by the immunohistochemical
method to determine the occurrence,
localization and distribution of
serotonin. Serotonin immunoreactive cells
(SIC) in the trachea were only solitary
cells; they were columnar, fusiform and
cuboidal in shape between and/or under the
ciliated cells, and they were identified
in all spheres of the trachea. Iinyea
cranial area developed sphincter, a large
number of SIC were seen. The number of SIC
in this area was about 5 per 100x100um?.
But their numbers decreased in the caudal
area of the trachea gradually, and non-
cilliated cells between capillaries, such
as the respiratory portion in the lung,
were seen in the dorsal part of the
trachea, SIC were not identified, and
these structures were located near the
lung. SIC corresponded to neuroendocrin
cells of the mammalian lung, they may be
paraneuron cells or APUD-type cells. These
cells, may be related to regulation of
hypoxia or other chemical and physical
stimulation. The sphincter-developed area
near the air entrance seemed to be related
to these regulatory systems.
THREE DIMENSIONAL POSITION OF THE
EPITHELIAL CELLS OF THE QUAIL LUNG
A.Kimura?, D.Adriaensen?, T.Gomi+, J-P.
Timmermans?, M.H.A.De Groodt-Lasseel?,
D.W.Scheuermann2, Y.Kikuchi*? and K.Kishi?
1Dept. of Anat. | Sch. soxeemed: Toho
Univ., Tokyo; 2Lab. of celle Bion and
Histol., Univ. Centre of Antwerp, Antwerp,
Belgium
Birds’ respiratory systems manage
breathing with an air sac; This differs
from the respiratory systems of mammals,
reptiles, and amphibians. A computerized
three-dimensional image processor and a
resin casting method were used to analyzed
the quail's airway system. An analysis of
the three-dimensional position of the
bronchi system was attempted. Location of
the epithelial cells of the lung were
further examined.
The divergence of the secondary
bronchi was chiefly exhibited on the
dorsal side by the resin casting specimen.
Moreover, a lot of anastomosis was
exhibited in the secondary and third
bronchi. By light and electron
microscope, two types alveolar epithelial
cells were found: Type II alveolar
epithelial cell groups, which included
OLBs, existed near the airway; moreover,
Type I alveolar epithelial cells, which
covered capillaries, were located in the
respiratory area. This was able to be
confirmed visibly through a three-
dimensional reconstructing imaging system.
Cell Biology and Morphology
SURFACTANT PRODUCING CELLS IN BRONCHIOLES
AND TERMINAL BRONCHI OF THE RAT LUNG.
T.Gomi?, D.W.Scheuermann?, D.Adriaensen2,
J-P.Timmermans2, M.H.A.De Groodt-Lasseel2,
A.Kimura!, Y.Kikuchi?, K.Kishi?.
1Dept. of Anat. Sch. of Med. Toho Univ.,
HokyoreesLab. of cell Biol. and Histol. ,
Univ. Centre of Antwerp, Antwerp (Belgium)
It is well known that the type II
alveolar pneumocyte is the main secretor of
alveolar surfactant. However, the site of
origin of the bronchiolar lining layer is
largely uncertain. Using a fixative
containing glutaraldehyde, paraformaldehyde
and tannic acid, an ultrastructural study
of all the cell types present in
bronchioles and terminal bronchi of the
adult rat lung was performed. In the
investigated area, the non-ciliated cells
(Clara cells) are dome-shaped and contain
large amounts of smooth endoplasmic
reticulum and mitochondria. The Clara cells
also harbour a variable amount of
osmiophilic lamellated bodies (OLBs). The
fine-structure of these OLBs appears
compatible with bimolecular leaflets of
lipoproteins. The OLBs are often aggregated
into large complexes and do not seem to be
membrane-bound. At the luminal surface of
Clara cells, images of OLBs suggesting
secretion are obvious. It can, therefore,
be postulated that the Clara cell plays a
role in the synthesis and secretion of
bronchiolar surfactant... Finally, it is
noteworthy that a small amount of inclusion
bodies, comparable to those found in Clara
cells, was also observed in ciliated cells
and in the rarely found goblet ceils of rat
lung bronchioles.
1291
1292 Behavior Biology and Ecology
ECOLOGICAL STUDY OF REPRODUCTION OF
Oulastrea cispata IN OKINAWA.
Y. Nakano! and K. Yamazato2 1Sesoko Marine Science Center,
Univ. of the Ryukyus, Okinawa, and 2Dept. of Biology, Col. of Sci.,
Univ. of the Ryukyus, Okinawa.
Oulastrea crispata, which is distinguished by a black
skeleton, lives patchily in depressions in shallow reef
flats or in turbid, muddy bays where few hermatypic
corals occur. No gonads were observed in polyps
before July, 1992, using a microscope, but gonads with
mature eggs and sperm were observed from July
onwards. Each polyp was hermaphrodite, with ovarial
and testicular regions in all twelve mesenterial
filaments. Eggs within the gonads were of different
sizes. Mature white eggs without zooxanthellae were
about 130 pm in diameter. Eggs were spawned from
midnight to dawn, sank to the bottom, and changed
into planulae without zooxanthellae before noon. The
spawning season was longer than in other faviid
corals. O. crispata continued to spawn eggs every few
days starting in July regardless of lunar phase. Another
type of planula was observed that was the same size or
smaller than mature eggs of Oulastrea. A few
planulae of this type, with zooxanthellae and
nematocysts, were released during the rest periods
between spawnings. These planulae may stem from
mesenterial filaments and represent a form of asexual
reproduction.
GROWTH RELATED MIGRATION OF THE INTERTIDAL
SNAIL, MONODONTA LABIO CONFUSA, WITH A
CONSIDERATION TO A RELATION TO THE FOOD
AMOUNT
A. lijima. Toho Univ., Chiba.
In the intertidal rocky shore at
Kominato, Pacific coast of Boso Peninsula
structure of the shore shows mozaic pattern
which consits of stable rock surfaces and
pachy cobble fields. Species composition of
the benthic organisms between two bottom
structures is quite defference. Molluscan
grazers are very abundant in both bottom
stuructures, but most species prefer either
bottom structure except the trochid snail
Monodonta labio confusa which very numerous
in the middle inter-tidal zone not only of
the stable rock surface but also of the
cobble field. It was observed by recapture
of marked snails that juveniles of the snail
inhabited in the cobble fields and migrated
to the stable rocks after it reached about
5mm in shell hight. The analysis of
chlorophyll amount of epilithic microalgae
showed the amount tended to be higher on the
stable and macroalgae-free rock surfaces. The
food amount may be an important factor for
the distribution of the Monodonta snail.
AGGREGATION FOR SPAWNING IN THE BREEDING
SEASON OF THE SEA STAR, ASTERINA MINOR.
H. Tominagat and M. Komatsu2. ‘Kanazu
High Sch., Fukui and ?Dept. of Biol., Fac.
of Sci., Toyama Univ., Toyama.
———
It is known that in the laboratory con-
dition the hermaphroditic sea star,
Asterina minor aggregates for spawning
(Komatsu et al., 1979). However, no de-
tailed observation has been made for the
spawning behavior in the natural habitat.
In the present study,field observation
was carried out using a 1 m2 guadrant at
Echizen Coast in Fukui Pref., During the
breeding season, aggregate behavior,
density and biomass were studied. Asa
result, it was revealed that (1) this sea
star bred between late May and early June,
(2) the size of maturity was more than 100
mg wet weight and 4 mm major_radius.
Furthermore, density (11.4/m“), biomass
(13114 mg) and the ratio of overlap (14.9
%) are large in the breeding season than in
the non-breeding season.
Judging from these results,it was sug-
gested that mature, large sea stars aggre-
gated just before the spawning, overlapped
each other for about 2 weeks, and then dis-
persed quickly after spawning. Self-fer-
tilization was confirmed by an isolated
experiment. This breeding behavior may
give a high rate of cross-fertilization for
a high degree of efficiency in breeding.
ORIENTATION AND POSITIONAL ARRANGEMENT OF
ZOOIDS IN FOUR DIFFERENT ASCIDIAN COLONIES.
Y. Taneda and T. Watanabe. Dep. of Biol.,
Fac. of Educat., Yokohama Natl. Univ. ,Yoko-
hama.
From the structural organization, ascid-
ian colonies are divided into four differ-
ent types. Polycitor proiiferus forms a
colony without both common vascular system
and common cloaca. Aplidium yamazii posse-
sses common cloaca, while does not common
vascular system. In <Symplegma reptans,
common vascular system is present, while
common cloaca is absent. In #Sotryllus sch-
losseri, common vascular system and common
cloaca are both present. Distances between
two adjacent branchial apertures were com-
pared among these four colonies, because
sufficient food supply is most important in
life of the animals. The distance between
two adjacent branchial apertures is approx-
imately constant, however differs from spe-
cies. The distance per size of zooid in P.
proiiferus is longer than that in 4. yame
ziz, and that in S&S. reptans is longer than
that in &. schlosserz. In a similar manner,
that in ?. projiferus is longer than that
in S§. reptans, and that ind. yamaziz is
longer than that in #8. schlosserz. Forma-
tion of common vascular system or common
cloaca may shorten the distance between two
adjacent branchial apertures, therefore, it
may increase the number of zooids per same
area of the colony.
Behavior Biology and Ecology 1293
COLONY FORMATION IN THE SERPULID, SALMACINA
DYSTERI (HUXLEY) (ANNELIDA, POLY CHAETA)
Eijiroh Nishi! and Moritaka Nishihira’, 1Amakusa Mar. Biol.
Lab., Fac. Sci., Kyushu Univ., Amakusa, and 2Biol. Instit.,
Fac. Sci., Tohoku Univ., Sendai.
In Okinawan coral reefs, many individuals of a tubicolous
polychaete Salmacina dysteri (Huxley) (Annelida, Serpulidae)
aggregate and form an arborescent colony on various
substrates. This species is protandrous or simultaneous
hermaphroditic, reproduce sexually and asexually, and broods
embryos in the tube until 3 setigerous stage. Internally fertilized
eggs are liberated within tubes and embryos develop in the tube
along compressed adult abdomen.
Frequent asexual reproduction seemed to contribute greatly to
colony formation. Asexually reproduced buds did not disperse,
but remained on the parent or neighbouring tubes. They regulate
distance from the nearest tube aperture during tube growth not
to interfere the branchial crowns of their neighbours, but not to
separated far from each other. This seems effective in
maintaining strength for fragile colonies.
Larvae, which are capable of dispersal, settled gregariously,
even On the colonies of the same species. Fusions of colonies
occurred commonly in the field. Starch-gel electrophoretical
analyses of 35 colonies showed intra-colonial variations in GPI
(Glucose-6-phosphate isomerase), MDH (Malate dehydroge-
nase) and IDH (Isocitrate dehydrogenase) loci on 10 colonies.
These evidences strongly suggest a possibility of colony
formation by multi-clonal aggregation. For this species, rapid
colony growth, even by joining with unidentical clone mates,
seems beneficial because survival rate of colonies and the
proportion of sexually reproducing worms increase with colony
size.
ORIENTATION OF INFANT JAPANESE TOADS SOON
AFTER METAMORPHOSIS DURING TERRESTRIAL
MIGRATION.
Y. Ooka!, S. Ooka? and S. Ishiil, !Dept. of
Biol., Sch. of Edu., Waseda Univ., Tokyo
and #Atomigakuen Jr. Coll., Tokyo.
Newly metamorphosed infants of the
Japanese toads, Bufo japonicus, migrate on
the ground to a direction away from the
pond where they have lived. To know the
mean to orient the direction, we caught
several iNividuals of the infants which had
landed or were going to land, put them ona
plastic board. After confirming their
moving direction, we rotated the board 180
degree horizontally at the site of their
landing and/or translocated to the opposite
side of the pond. all these treatments did
not affect the original direction of the
movement. In the other words, rotated toads
turned around and went away from the pond,
and translocated toads with or without
rotation moved toward the pond. When we
covered moving infants with a plastic
column painted black, they could not orient
a certain direction. When we did it with a
transparent plastic column, they retained
the original direction. These experiments
strongly suggest that newly-metamorphosed
toads orient to the direction away from the
pond by means of the visual sense and not
by using the magnetic field of the earth, a
wind direction, or some cue from the pond,
while adult toads have been reported to use
the olfactory sense to orient to the
breeding pond during the breeding season.
COMPARATIVE ANALYSIS OF COMMUNICATION WITH
URINATION IN TWO JAPANESE APODEMUS SPE-
CIES.
M.Daumae. When Cre ihi@llo, (Gfopilibe of Arts
and Sci., Univ. of Tokyo, Tokyo.
The urination pattern and the response to
urine of small Japanese field mice (A.
argenteus) and Japanese field mice (A.
Speciosus) were analyzed. From my previous
reports, the two species differed in urine
marking patterns when they were exposed to
novel environment. To see if A. argenteus
and A. Speciosus have the ability to
distinguish between male and female urine,
animals were exposed to both sex type
urine depositions. In A. speciosus both
male and female urinated around female
urine. Although in A. argenteus, female
mice did not show significant difference
in time spent around urine and _ urination
patterns, male mice preferred female urine
to male. But when blank area was taken as
control to male urine or female urine,
male mice did not show significant prefer-
ence. Also when they were exposed to both
sex type urine, they preferred blank area.
This result suggests that male mice avoid
male urine presented with female urine.
This complicated response to urine of A.
argenteus can be correlated to territori-
ality and their mating system which differ
from A. speciosus.
THE RELATIONSHIP BETWEEN THE OCCURRENCE OF PATER-
NAL BEHAVIOR AND ANDROGEN IN MICE (MUS MUSCULUS)
F. Matsumoto and T. Kimura. Dept. of Biol., Coll.
of Arts and Sci., Univ. of Tokyo, Tokyo.
Male mice which had copulated and cohabited
with the mate until her delivery showed various
paternal behavior patterns with much higher fre-
quency than naive males. We already reported that
the experience of copulation affected the frequen-
cy of the occurrence of paternal behaviors. In
order to study the physiological factor which
affects the paternal behaviors, first of all, we
examined the relationship between androgen
secretion and the occurrence of paternal behav-
iors.Most of naive adult male mice which had
castrated at 13-14 weeks of age and had recovered
from the operation at least for 3 weeks, behaved
parentally. While naive adult male mice which had
castrated and received subcutaneous injections of
50ug testosterone propionate every day from the
operation for 3 weeks, tended to kill or ignore
the pups. These indicate that androgen seems to
restrain the occurrence of paternal behaviors.
In the next experiment, serum testosterone
levels were determined in sexually naive male mice
at 10 weeks of age and in the same mice which
thereafter copulated and cohabited with the mate
until her delivery("experience”). The serum tes-
tosterone levels in the “experience” mice which
behaved parentally showed a tendency to decrease
than in the same mice at naive state.
It is highly probable that the “experience”
causes androgen depletion, which enhances paternal
behaviors. Further studies are needed to clarify
the crucial factor, among those involved in the
“experience”, which causes this hormonal change.
1294 Behavior Biology and Ecology
EFFECTS OF CAGE-MATES ON SOCIAL ORDER OF
MALE MICE.
S. Hayashi. Dept.Biol., Fac.Educ.,
Kagoshima Univ., Kagoshima.
Male mice were raised with one male
cage-mate ( 2-male group ) or two male
cagemates ( 3-male group ) from 4 to 13
weeks of age. They were transferred to a
new cage every 7 days. Immediately after
renewing cages, the mice were observed for
30 min to assure the dominant-subordinate
relationships among cage-mates. Pairing
commenced at 13 weeks of age. Each male
was paired with another male of the same
group every 3 days. The males of 2-male
and 3-male groups were paired with 14 and
15 males, respectively. The pairing two
males were separatd by a wire net barrier
in a cage for 1 day and then the barrier
was removed for 30 minutes. The frequency
of the aggressive behavior during the last
20 minutes was analyzed.
The order of the mice was estimated by
the results of accumulated matches and
attributed to dominant and subordinate
males respectively.
The results showed that the order of
the dominant male was correlatd to that of
the subordinate cage-mate in both groups.
When the dominant male was in high rank
comparing to dominant males of other
cages, the cage-mate which was subordinate
one was also apt to be in high rank among
subordinates.
BEHAVIORS OF THE BENTHIC ARROW
WORM (PARASPADELLA GOTOI, CHAETO-
GNATHA) IN RESPONSE TO WATER FLOW.
T. Goto and T. Takagi, Dept. Biol., Fac. Educ.,
Mie University, Mie
Arrow worms are carnivores which feed on cope-
pods and fish larvae. They attack and bite prey that
approaches within a few mm. It has been reported
that arrow worms attack a fine probe when it is
vibrated at appropriate frequencies, leading to
speculation that they recognize the specific vibra-
tion rates produced by swimming copepods and
hence can select their food. However, reexamina-
tion of this experiment has not been successful. We
thought that water flow caused by a vibrating probe
may be important for the feeding and examined
responses to water flow stimuli using a benthic
arrow worm Paraspadella gotoi. Stimulus was
given from a jet nozzle (300 wm in diameter)
connected to a compressed sea water tank. A single
stimulus of 1 cm/s water velocity elicited a feeding
response that is characterized by expanding their
grasping spines. Stronger stimuli induced an escape
reaction and reduced feeding response. Repetitive
stimuli of low velocity water flow also elicited a
feeding response to each stimulus, indicating that
water flow is an essential key stimulation for induc-
ing feeding response in P. gotoi.
FEMALE RECOGNITION BY THE MALE
CRICKET GRYLLUS BIMACULATUS
J.Nagamoto and M.Hisada. Zool. Inst., Fac.
of Sci., Hokkaido Univ.,Sapporo.
A Gryllus bimaculatus male responds
with the courtship song to females and
with the aggressive song to males when
he encounters the conspecifics.
We examined what sensory cue or cues
are required in the sex recognition.
Males made the sexual discrimination
towards the antennae severed from
other individuals when they were
artificially palpated in the antennae
or the palpi.
Chemoreception was essential to
female recognition. The polyethylene
fiber that was smeared with the
substance(s) on the female cuticle
surface could elicit courtship
behaviors.
The effective substance(s) seems to
be a moderately volatile hydrocarbon.
Female antennae treated with hexane or
acetone failed to release courtship
behaviors. Antennae lost their ability
3-4 hr after they were detached from a
female. However,the efficacy was lost
by treatment with EtOH. This suggests
that the effective substance(s) could
be a polar compound.
FOOD DISTRIBUTION DEPENDENCE IN THE FRACTAL
FEEDING BEHAVIOR OF DROSOPHILA
H. Tanigawa!, I. Shimadal, Y. Hayakawa2,
and H. Hara
Dept. Biol. Sci., 2Res. Inst. Elec. Com.,
3Eng. Sci., Fac. Eng., Tohoku Univ., Sendai.
With the aid of our automatic analysis
system for the feeding behavior of Droso-
hila we obtained a huge data of dwelling
time on food in paired dense (60 wells) and
sparse (8 wells) food distribution experi-
ments.
The sparse food distribution dwelling
time is not only much longer than the dense
one but also has a tendency to produce an
exponential distribution of dwelling time.
During one hour of feeding it shows a
transition from a fractal, that is an in-
verse power law distribution of dwelling
time to non-fractal exponential. In the
choice situation between 100 mM (100S) and
10 mM sucrose (10S) in the dense food dis-
tribution, the dwelling time on 10S shows
an inverse power distribution. The dwelling
time on 100S, on the other hand, gives a
distribution close to an exponential. In
the sparse food distribution, more striking
difference between 100S and 10S is observed
with the same tendency. Under these situ-
ations, flies feed exclusively on 100S.
These suggest an important biological mean-
ing in the fractal feeding behavior of
Drosophila.
Behavior Biology and Ecology 1295
FURTHER INVESTIGATION ON CIRCADIAN RHYTHM
OF THE CLOCK MUTANT, TOKI.
A.Matsumoto, K.Tomioka and Y.Chiba. Biol.
Inst., Yamaguchi Univ., Yamaguchi.
A screening for circadian rhythm mutants
from one thousand strains of Drosophila
melanogaster with EMS-treated second
chromosomes resulted in the isolation of a
new semidominant mutant, Toki. This muta-
tion altered four circadian parameters:
rt, #, @/p and an activity level, which
show larger value than those of wild type
In the present study, we examined the ap-
plicability, to Toki, of Aschoff’s circa-
dian rule that, in the day-active animal,
the free-running period is shorter in
higher light intensity. As the result, it
was found 1) that the free-running rhythm
was lengthened as light intensity became
higher in Toki and the wild-type, violat-
ing the circadian rule, and 2) that Toki
waS more sensitive to the change of light
intensity so that, under the constant
light, the damping occurred at 0.1 lux
whereas it occurred at 1 lux in the wild-
type. We also investigated the genetic
interaction between Toki and the other
cloek mutations on the X chromosome (per,
per“ and And). Toki affected them such
that the free-running period is lengthened
and, when entrained to 24 h LD cycle, the
activity offset becomes to be timed earli-
er: such a relation between the period and
the timing of the activity offset is
Opposite to that generally known
SPONTANEOUS DESYNCHRONIZATION OF MULTIPLE
CIRCADIAN COMPONENTS IN LOCOMOTOR/FLIGHT
ACTIVITY OF THE MOSQUITO.
Y. Chiba, S. Takeda, A. Matsumoto, and
K.Tomioka. Biol. Inst., Yamaguchi Univ.,
Yamaguchi, 753 Japan.
We reported in 1990 that a strain of
Culex pipiens molestus might have two
circadian components in the activity which
differ in sensitivity to light intensity,
as has been postulated in rodents (Pitten-
drigh and Daan 1976) and the other species
of mosquito (Clopton 1984).
Recently we demonstrated that two osilla-
tions existed more conclusively than any of
the above-mentioned cases, using C. p.
pallens held in DD. One oscillation has a
period of 22.5 h, and the other the longer
period of levels of 24h in male and 23 h
in female.
These findings are significant, contrib-
uting to understanding of two aspects of
the entrained circadian pattern: the
diphase and the insemination dependency
That is, in male, the two circadian compo-
nents were apparently diphasic in DD,
giving rise to a question as to whether the
evening and the morning peaks are underlain
really by separate oscillations as have
generally been postulated. On the other
hand, it was preliminarily observed that
the longer component was intensified in the
inseminated female to yield the insemina-
tion dependent change of circadian pattern.
RESPONSE OF SWIMMING ACTIVITY RHYTHM OF THE
CRUSTACEAN DIMORPHOSTYLIS ASIATICA
(CUMACEA) TO SINGLE LIGHT PULSES.
T. Akiyama. Ushimado Marine Laboratory,
Okayama University, Okayama.
The crustacean Dimorphostylis asiatica
exhibits nocturnal swimming activity which
coincides with high tide. Although day-
night synchronization of the rhythmicity in
the field was obvious, free-running ac-—
tivity pattern under constant darkness in
the laboratory did not show the evidence
that endogenous activity rhythm of the
animals can be synchronized with solar day
cycle.
In order to investigate whether en-
dogenous pacemaker of D. asiatica is
responsive to the light stimulus, freshly
collected animals kept under DD condition
were exposed to single 4h light pulses. The
pulses were given on 4-5th day when most of
the animals exhibited free-running bimodal
(circatidal) rhythms. Most of calculated
phase shifts on 29 animals was shorter than
l hour. Direction (advance or delay) of
the phase shift seemed to have no correla-
tion with the circadian time when the light
pulse was imposed, These results suggest
that the endogenous pacemaker which con—
trols bimodal activity pattern of D.
asiatica does not sense the light stimulus.
EFFECT OF WAVELENGTH ON THE ENTRAINMENT OF
LOCOMOTOR ACTIVITY RHYTHMS IN THE JAPANESE
GRASS LIZARD (Takydromus smaragdimus)
M. Ohtani, and T. Oishi, Dept. of Biology, Nara Women's
Univ., Nara 630
Locomotor activity rhythm of Japanese grass lizards can
be entrained by both light-dark cycles and temperature
cycles. In order to estimate photoreceptor pigments
involved in the entrainment by light-dark cycles, we
performed (1) an experiment to find action spectra and
(2) immnocytochemistly by use of anti-serum against
bovine rhodopsin (Rh-As) and monoclonal antibodies
against chicken iodopsin (lo-mAb).
(1) The lizards were exposed to monochromatic light
(400,450,500,550,600,650,700nm) and infrared
radiation (800nm~) by using interference filters under the
light phase of LD cycles. The number of photons were
adjusted to be the same (5X10'3 photons/cm2) for
different wavelengths.
Locomotor activity rhythm of the lizards were entrained
to 400 - 700nm of visible light. But infrared radiation
was not effective for the entrainment.
(2) The photoreceptor cells in the eye were stained by
both Rh-As and lo-mAb. Some of the pinealocytes were
stained by lo-mAb, but all of the pinealocytes were
immno-negatived to Rh-As. The cells in the parietal eye
were not stained by either Rh-As or lo-mAb. Thus, the
composition of visual pigments in each photoreceptor
organ was different, which suggests different function for
each organ.
1296 Behavior Biology and Ecology
DIURNAL CHANGE OF BEHAVIOR PATTERN IN THE
CHINESE MUDSKIPPER, Periophthalmus cantonensis
Y.Ikebe, and T.Oishi, Dept. cf Biology,
NairalWomenWissU natvae as Ng ns
The Chinese mudskipper is an amphibious
fish and inhabits tideland. In the tide-
land of Utinoura Bay, Wakayama.,the fish
first appears on the mud flat in April.
The number of fish on the tideland increase
from April to June and levels off
thereafter. We investigated seasonal and
diurnal changes of the behavior pattern in
the mudskipper.
Four types of behavior (Comfort,
Feeding, Nesting and Courtship) were
classified and the number of fish
exhibiting each type of behavior was
counted every 30 minutes from 10:00 to
18:00 for about 4 days in each season.
In April and May ( non-breeding season),
only feeding behavior was observed and the
fish fed continuously during low tide and
stopped feeding about 30 minutes before
rising tide. From June to August, nesting
and courtship behavior (reproductive
behavior) were observed in addition to
feeding behavior. The reproductive
behavior was observed throughout census
bouts in a day.
Both high and low temperature seem to
decrease the number of fish appeared on the
mud flat and the feeding activity.
However, the reproductive behavior was not
affected by temperature.
DIURNAL AND ANNUAL CHANGES IN THE NUMBER
OF LOACHES CAPTURED IN THE FIELD.
M. Naruse and T. Oishi, Dept., of Biol.,
Fac., of Sci., Nara Women's Univ., Nara.
In order to investigate the diurnal and
annual locomotor activity rhythm of
loaches (Misgurnus anguillicaudatus) in
the open air, we have caught loaches
regularly by traps settled in small
streams along the rice fields near Nara
city for two years since 1990.
Loaches began to appear in April and
disappear in October or November in the
streams. In winter, loaches seemed to
hibernate in this field. After the
appearance in April, only a small number
of fish was caught in May and June. This
season is supposed to be the reproductive
season for loaches as the high level of
gonadosomatic index (GSI) indicates. In
this season, most of adult fish seem to
enter and stay in the rice fields to
breed.
The number of loaches generally seemed
to be greater in the dark phase than in
the light phase throughout the year
especially at the time when large numbers
were recorded. The tendency of change in
the number of fish did not differ between
sexes or body sizes. When we examined the
relation between the environmental factors
(day length and water temperature) and
diurnal change in the number of fish
captured, both day length and water
temperature seemed to be involved in this
change.
Taxonomy and Systematics
THE FAUNA OF GREGARINES IN SOUTH INDIA
K. Hoshide Biol. Lab. Fac. of Educ.
Yamaguchi Univ., Yamguchi
The fauna of gregarines is studied at
Gulbarga and Calicut in south India.
Five species of septate gregarines,
Stylocephalus depressicus, S. punctula-
tus, Cystocephalus rhytinotus, Xiphoceph-
alus gonocephali, X. reitteri were ob-
served from five species of coleoptera
beetles at Gulbarga. One species of
septate gregarine, Leidyana sp., was
observed from Lepidoptera larva at Cali-
cut. Lots of gamonts and cephalines of S.
depressicus were obtained. They have a
characterlistic epimerite. The epimerite
is lance-shaped which consits of long
slender cylindrical neck and flame-like
cone. The fine structure of the epimerite
was studied by SEM. and TEM. The gameto-
cysts and oocysts of S. depressicus were
also obtained. The spherical gameto-
cysts were kept in the moist chamber for
3 days and the hat-like oocysts were
dehiced in chain by simple rupture. The
effects of digestive juce for releasing
the sporozoites were examined. The sporo-
zoites were released from oocysts after 2
to 3 hours to put the oocysts in the
digestive juce of the host.
FAUNISTIC CHANGES OF THE ROTIFERA IN THE
COASTAL PREFECTURES.1.TOTTORI & SHIMANE-KEN
M.Sudzuki'and K.Fukuta.Biol.Lab.,Nihon Univ
Omiya and? Biol.Lab.,Tottori Univ. ,Tottori.
Followings have been found in the samples
(28-29/VIII'90) from such mixohaline(mh)/fr
eshwater(fw) lake,ponds,castle moat & river
as Naka-umi(N=mh) , Ohashi-gawa(0O=mh) , Shinji-
ko(S=mh) , T6g6-ike(TG=mh) , Tanega—ike(TN=mh) ,
Koyama—ike(K=mh) ,Mizushiri-ike(M=fw) ,O-hori
(C=fw) & Daigaku-ike(D=fw) in order of sali
nity:B.r. decemcornis(N),B.r.estoniana(N,QO),
Rotaria | sp.(0),Tchoc.c. chattoni(0O, C)Cephalo
della sp.(S,M),An.f. urawaensis(S, TN)Pu.m.mu
sicola(TG), Philodina sp. (TG) ,Con.coenobasis
(TN,K,C),C.unicornis(TN,C),C. mutabilis(TN,&)
M.b.bulla(TN,M,C),M.b.dentata(TN,K),M.clost
erocerca(TN), Lec.c. curvicornis(TN), bye Pp- pate
iIla(TN), ost; obtusa(TN,C) ,E. lyra(TN), (E. l.mye
rsi(K), E.dilatata(K), Tpl. plicata(TN), {K. .coch
Tearis(TN,K,C,D), K.v.tropica(TN,K,M,C,D),C.
Ovalis(TN,K), Tchoc. Capcina(TN, D),D. similis
(TN, M) ,Po.t.vulgaris(TNK,C) ,H.fennica?(TN,®)
Asp.p. herricki(TN) , A. lee .priodonta(K) , Lec.hor
nemani(K), L.luna(K) ,M.stn.lineata(K), 7 Tchoc.
elongata(K), M. See meeee ee Asp.sieboldi
(K,M),Te.p- trilobata(K), Eat quadricornis(K),
Tchot. tetractis(K),F. Ae mystacina(K, M,C,D)B.
a.angularis(K,M,C), s. diversicornis(K, M),S.d
homoceros(M) ,B. calyciflorus(K, M,C,D)B.c. bor
gerti(M),B. falcatus(K),B.f.f. reductus(C,D),
B.a.bidens(D),B. budapestinensis(M) , B.forfic
Bae (G4 1D))) iB. q. brevispinus(M) , IPO) 8 « eeigiay
oy Siena Setioy, T. pusilla( Cc), Lilifero-
trocha sp.(C),Pom. sulcata(C),Lophocharis © n.
sp.(C),Asc.s. indica(M), B.a.pseudokeikoa(M) .
1297
URBAN TARDIGRADES IN THE TOHOKU AREA.
K.Utsugi. Dept. of Biol.,Tokyo Women's
Med. College, Tokyo.
To study the Japanese tardigrades,mosses
or lichens were collected from 88 stations
of 6 cities in the Tohoku area (Aomori, A-
kita, Morioka, Yamagata,Sendai and Fukushi-
ma) during a period from 1984 to 1992.
The tardigrades were collected from the
Macerating samples. After treated slightly
with heat, the samples were mounted with
Gum-chloral. The presence of the animals
were microscopically examined. Some of them
were observed by SEM.
13 species were found at 23 stations in
6 cities. 3 species belonging to Eutardi-
grada(Macrobiotus harmsworthi, M.hufelandi
and Milnesium tardigradum) were commonly
distributed in many stations. However,the
other 6 species of Eutardigrada (M.hufel-
andioides, M.intermedius, M.richtersi, Hyp-
sibius baumanni, Esohypsibius reticulatus
to Heterotardigrada (Echiniscus baius, E.
canadensis, E.japonicus and Pseudechiniscus
facettalis) were rarely found in urban
areas. It appears that the distribution
of Heterotardigrada are scarce in the To-
hoku area ; especially E,japonicus which
was distributed widely in many cities in
southern Japan, but was found at only one
station in Morioka. In addition, we can
determine that since the common species of
tardigrades were not found in Aomori, they
do not exit in this region. Only I.reticu-
latus were obtained from one station out
of 26.
GENUS DIPHASCON (TARDIGRADA) FROM JAPAN.
M. Ito! and K. Utsugi-. lInsect Taxon.
& Ecol. Lab., Forest Biol. Div., Forest-—
ry & Forest ‘Products. Res. Inst., Kuki-
zaki and “Dept of Biol., Tokyo Women’s
Med. Coll., Tokyo.
ite Terrestrial tardigrade fauna of
Japan has been surveyed since 1984. In
the course of survey, 15 species of
genus Diphascon PLATE, 1889 were found
from soil, moss and lichen samples of 17
areas (including various types of habi-
tats). Tardigrades were sorted directly
Or extracted by Baermann funnels from
the samples and observed under a Nomar-—
ski’s differential interference micro-
scope after mounted on slides.
an Among them, 12 were known species.
Two species (D. pingue & scoticum) were
already recorded by some historical
workers. We added remaining 10 species
as the first records from Japan. These
are listed as follows: D.(D.) higginsi,
nobilei, oculatum, patanei, recamieri,
rugosum; D.(A.) belgicae, Carolae,
Mariae, prorsirostre.
3. Three species of genus Diphascon are
new to science. One of them has two
drop-like thickenings. It is a inter-
esting discovery in phylogeny of the
genus Diphascon.
4. In general, species of Diphascon
prefer to surface soil and they were
very rare in moss and lichen. ine WS
clear that Diphascon become dominant in
full matured forest soil. Therefore,
the genus seems to be very useful as an
indicator of forest environment.
1298 Taxonomy and Systematics
KARYOLOGICAL AND TAXONOMIC STUDIES OF THE
DUGESIA SPECIES IN SOUTHEAST ASIA. XVIII.
CHROMOSOMES OF JUVENILES HATCHED FROM CO-
COONS OF DUGESIA JAPONICA RYUKYUENSIS,
S.Tamural, I.0ki* and M.Kawakatsu3. 10sa-
ka Pref.Inst.Publ.Health, Osaka, 2OEPA,
Osaka, 3Fuji Women's College, Sapporo.
Numerous variations of karyotypes are
known in both subspecies of Dugesia japo-
nica. In D.j.ryukyuensis from Okinawa Is-
land, new karyological data were obtained.
1) Karyotypes of 19 juveniles hatched from
5 cocoons (Kinché and Ginowan-1 popula-—
tions) were: diploid (2), diploidy (6),
triploidy (11). Karyotypes of juveniles
hatched from each cocoon showed 4 combina-
tions: i) diploidy only (1 cocoon), ii)
triploidy only (2 cocoons), iii) diploidy
& triploidy (1 cocoon), and iv) diploid,
diploidy & triploidy (1 cocoon). There-
fore, juveniles hatched from one cocoon
can all be homogeneous sharing one karyo-
type, or heterogeneous showing different
karyotypes.
2) One triploidie specimen (P) of the Kin-
ché population cultured under isolated con-
ditions reached maturity and laid several
cocoons. Karyotypes of 4 juveniles (F,)
from a single cocoon were: triploidy (4),
mixoploidy (1). One triploidic juvenile
cultured under isolated conditions reached
maturity and laid cocoons. A single juve-
nile (F2) from one of these cocoons had a
triploidy karyotype. These data may be ex-
plained by parthenogenesis and/or unusual
meiotic divisions or cleavage of the egg.
SYSTEMATICS AND COMPARATIVE MORPHOLOGY OF A
NEW SPECIES OF POLYSTYLIPHORA (TURBELLARIA,
PROSERIATA, POLYSTYLIPHORIDAE) FROM JAPAN.
K.-I. Tajika. Dept. of Biol., Nihon Univ.
Sch. of Med., Itabashi, Tokyo.
The marine interstitial genus Polystyli-
phora Ax, 1958 is characterized by numerous
uniform prostatoid organs that are serially
arranged caudal to the male copulatory
organ. Each of the prostatoid organs is
furnished with a species-specific, funnel-
shaped stylet of unknown function. Since
1983 a limited number of specimens belong-
ing to the genus have been collected from
Misaki, Johga-shima, Monkey Is. (central
Japan), and Ishigaki-jima (southwestern
Japan). Analysis of their prostatoid
organs reveals that the number of the
prostatoid organs attains to 46 and the
stylet does not differ in shape among the
specimens from these different localities.
Furthermore, the stylet of the male copula-
tory organ as well as the prostatoid stylet
is clearly distinguished from that of six
known species. Discovery of the completely
matured specimens from Monkey Is. makes it
clear that many testes always lie beneath
voluminous vitellaria in the prepharyngeal
body. A pair of seminal vesicle sometimes
communicate transversely with each other.
Many (maximum 16) ovaries occupy a strictly
lateral position of the body, in contrast
to cases reported so far on the Nematopla-
nidae. In relation to this, it is diffi-
cult to justify such an idea that the genus
should be included in the Nematoplanidae.
MARINE NEMATODES OF THE GENUS EURYSTOMINA
FILIPJEV, COLLECTED FROM HOKKAIDO, JAPAN.
Ke Katto Dept. Biol., Sapporo Medical
College, Sapporo.
Three species of the Eurystomina have
been found around Hokkaido, northern Japan.
One of them was identified with 1
ophthalmophora (Steiner), known from
Shirahama, central Japan. The remaining two
are new species. E. sp. 1 is characterized
by the excretory pore opening before
cephalic setae, the ocelli located at 3.5-
4,3 buccal cavity length from anterior, the
spicules with smooth ends, the gubernaculum
with long apophysis slightly bending
dorsally, and two precloacal supplements
with developed wings. E. sp. 1 is similar
to E. parva Yoshimura, from Okinawa,
southernmost Japan, among the species
having a gubernaculum with long apophysis
but differs from it in the features of the
body length, de Man’s ratios, and male
copulatory organs. E. sp. 2 is
characterized by the excretory pore opening
behind cephalic setae, the ocelli located
at about 2.0 buccal cavity length from
anterior, the stout gubernaculum with broad
and well cuticularized apophysis, and two
precloacal supplements with developed
wings. E. sp. 2 resembles E. indica
Micoletzky, known from Okinawa, among the
species having a stout gubernaculum but it
is clearly distinguished from the latter by
the feature of the precloacal supplements.
DIFFERENCES IN PARAGNATH NUMBER
BETWEEN TWO SIBLING SPECIES IN THE
BRACKISH-WATER POLYCHAETE,
"NEANTHES JAPONICA".
M. Sato. Dep. of Biol., Fac. of Sci.,
Kagoshima Univ., Kagoshima.
Our previous studies have suggested
that Neanthes japonica (Izuka, 1908) (Nereididae) is, in
fact, a complex of two sibling species (the small- and
large-egg species), which differ in reproductive and
developmental modes, karyotypes, setal morphology
in mature adults and electrophoretic patterns for some
enzyme loci. The paragnath number on each of areas I
to VII-VIII of a proboscis was assessed in 187 adults of
the small-egg species collected from 11 localities and
175 adults of the large-egg species from 9 localities in
Japan. Marked geographic variations in paragnath
number were observed even within a species. In 4
rivers in Kagoshima, where both species occurred, the
large-egg species tended to have more paragnaths
than the small-egg species except in area VII-VIII: The
two species in these rivers were almost completely
separated by paragnath number in area II. Cultured
worms from fertilization in laboratory were also
examined. The ratio of paragnath number in each area
of the cultured worms approximated to that of the
field population of the same species. It suggests a
genetic background for the differences between the
two species.
Taxonomy and Systematics 1299
GENETIC VARIATION AND DIFFERENTIATION IN TWO LOCAL
JAPANESE POPULATIONS OF THE SEA URCHIN, ASTHENOSOMA
IJIMAI : ELECTROPHORETIC ANALYSIS OF ALLOZYMES.
T. Minokawa?, S. Amemiya’, and N. Matsuoka?
‘Misaki Marine Biological Station, Fac. of Sci., Univ.
of Tokyo, Miura. *Dept. of Biol., Fac. of Sci.,
Hirosaki Univ., Hirosaki.
The sea urchins of family Echinothuriidae are unique
because of their special habitat in deep-sea water and
their peculiar developmental pattern with the
non-feeding floating larvae. The poisonous sea urchin,
Asthenosoma ijimai which is distributed along the south
coast of Japan from Sagami to Kagoshima Bays and the
west coast of Kyusyu is a common Japanese species of the
family.
As a first step of the biochemical systematic studies
of the echinothurioids, enzyme variation and
interlocality genetic differentiation were examined in
two local Japanese populations from Sagami and Kagoshima
Bays of these a urchin by means of gel electrophoresis
of 10 different enzymes. The avarage heterozygosity per
locus (H) was 26.3% and 29.5% for the Sagami and
Kagoshima populations, respectively. These values are
considerably higher than those reported in many other
echinoderms from shallow water, but comparable to those
from deep-sea water. The Nei’s genetic distance between
two local populations of the species was 0.087.
The value is higher than the values observed between
conspecific local populations of other echinoderm
species. It shows a certain degree of genetic
differentiation between the two local populations of the
species. From the allozyme study, it may be considered
that the speciation in the two populaions is now under
way.
THERMOSENSITIVITY OF SEA URCHIN EMBRYOS IS
DETERMINED MATERNALLY
H. Fujisawa, Fac. Educ., Saitama Univ., Urawa.
The sea urchin, Hemicentrotus pulcherrimus, is one
of the most widely distributed echinoids in Japan,
from the southern part of Hokkaido to Kagoshima.
This sea urchin spawns largely in the same season,
from winter to spring, regardless of difference in
habitat. The seawater temperature during the
spawning season is about 6° in Mutsu Bay, while
it is about 17°C in Sendai, Kagoshima. The differ-
ence in seawater temperature during the spawning
season is thus more than 10°C between the two
localities. In this study two groups of the sea
urchin were used. One group was collected in Mutsu
Bay and the other off the coast of Sendai, Kago-
shima. The embryo thermosensitivity in the two
groups was examined. Thermosensitivity is defined
as the temperature at which a sea urchin embryo is
able to develop normally. Thermosensitivity of
embryos of the sea urchins in Mutsu Bay was from
5 to 19°C, while that of the sea urchin in Sendai
was from 8 to 22°C. The limits of thermosensi-
tivity were found to be shifted significantly by
about 3°C. Next the thermosensitivity of the eggs
of sea urchins from Mutsu Bay (N(2)) fertilized
with the sperm of sea urchins from Sendai (S(<))
as well as that of the reciprocal cross (N(o')
x S(¢)) was examined. The thermosensitivity of
the (N(?)xS(o')) embryo was same as that of the
(N(2)XN(o")) embryo. The thermosensitivity of
the (N(o')X S(2)) embryo was also same as that of
the (S(9)xS(o)) embryo. This result suggests
that the thermosensitivity of sea urchin embryos
is maternally inherited.
DEVELOPMENT OF AN APODOUS HOLOTHUROID OF THE
GENUS, LABIDOPLAX.
M.Komatsu, T.Moritaki’, N.Suzuki2 and C.Oguro3.
Dept. of Biol., Fac. of Sci., Toyama Univ.,
Toyama, IToba Aqua., Toba, 2Noto Marine Lab. ,
Kanazawa Univ., Ishikawa, ~Toyama Univ.,Toyama.
Spawning in the laboratory occurs next
morning after collecting from about 25m
depth in Tsukumo Bay, Ishikawa on December
11, 1991. This holothuroid is hermaphro-
ditic and natural self-fertilization takes
place with sperm and eggs. Ova are orange
in color and 200pm in average diameter.
Four and half hr after spawning they are in
8-cell stage at 15°C. Cleavage is radial
and equal and resulting blastula is holo-
blastic. Gastrulation begins by invagina-
tion approximately 10 hr after spawning.
Embryos develop into a doliolaria with 4
transverse ciliary bands 2 days after spawn-
ing. They are ca. 400pm in length and 250m
in width. One day later, formation of cal-
Careous rings is apparent. Five primary
tentacles are visible showing the onset of
the pentactula stage. At this stage ciliary
bands become to degenerate. Around half a
month, pentactulae stop swimming and sink to
the bottom, signaling the completion of meta-
morphosis. Metamorphosed juveniles move the
substratum by using the tentacles. They are
about 400m in length and 180m in diameter.
In 2 species of Labidoplax, development
was reported; L. buskii and L. digitata.
The former undergoes the direct type of de-
velopment and the latter the indirect type.
Present study shows that development of the
present species is direct like L. buskii.
DEVELOPMENT OF THE SEA STAR, LUIDIA
MACULATA MULLER ET TROSHEL.
M. Komatsu, M. Kawai, S. Nojima’ and
C. Oguro? . Dept. of Biol., Fac. of Sci.,
Toyama Univ., Toyama, ‘Aizu Marine Lab.,
Fac. of Sci., Kumamoto Univ., Kumamoto
and * Toyama Univ., Toyama.
The entire process of development from
eggs to juveniles in the sea star, Luidia
maculata was observed.
The eggs are 173ym in average diameter.
One and half hours after fertilization,
the first cleavage occurs at 20CT. The
cleavage is total and radial. Embryos
develop into a bipinnaria through the
wrinkled blastula stage.
Metamorphosis takes place gradually at
the posterior portion of the bipinnaria
40 days after fertilization. At this stage
9 spicules, corresponding to the terminal
plate of adult skelton, appear. One week
later, bipinnariae reach a length of 2.5
mm.
Sixty-four days after fertilization,
metamorphosis is complated and resulting
juveniles are about 700ym in diameter
They have 9 arms and bear 2 pairs of tube-—
feet in each arm.
Development of the present species is
the non-brachiolarian type as all species
of Luidia previously reported. The present
observations show that just metamorphosed
juveniles have 9 arms like the adult. It
is very interesting that the time of arm
formation in multiarmed species of aster-
oid seems to be related to the systematics
of Luidia species.
1300 Taxonomy and Systematics
HAPLOID AND DIPLOID CELLS ARE BIOLOGICAL
ENTITIES BELONGING TO TWO DIFFERENT LEVELS
OF COMPLEXITY.
M. Dan-Sohkawa. Dept. of Biol., Fac. of
Sci., Osaka City Univ., Osaka.
Functional haploidy and diploidy of eu-
karyotic cell are defined not by the number
of chromosomes contained but by sexual
statuses. This statement is justified by
the abundant examples of parthenogenetic
and polyploid species found among wide
range of organisms.
Haploid and diploid cell, thus function—
ally defined, are evaluated in terms of
relative complexity of entities comprising
natural substances in a hierarchic order.
Based on the general rule underlying the
relation between such entities, two crite-
ria are set for the evaluation. (1) Is
diploid cell made of haploid cell? (2) Is
there any new function belonging to diploid
cell which is not found in haploid cell?
The answers are: (1) diploid cell is made
from two haploid cells through the process
of conjugation, and (2) the functions newly
acquired by diploid cell are the mechanism
of meiosis and the ability of cell differ-
entiation.
Fulfillment of the two criteria indi-
cates that diploid cell is one level more
complex than haploid cell.
This conclusion, suggests the Kingdom
Protista to be divided into two groups,
each of which containing those organisms
with and without sexual reproduction.
"“MULTI-CELL-TYPE MOSAIC HYPOTHESIS" ORIGIN OF MULTI-
CELLULAR ORGANISMS
T. Shinozawa, Fac. Eng., Gunma Univ., Kiryu, Gunma.
How does one explain the presence of the cilliary
structure, an organ for movement, in the vertebrate
photoreceptor cell as well as the olfactory cell ?
Why does the bacteria show chemotaxis to neurotran-
smitter, for example; amino acids, in the vertebra-
te neuron ? Finding an interfelon-like protein in a
plant, tobacco, is also surprising. Serial endosym-
biosis theory and recent accumulation of nucleotide
sequence information in many proteins still do not
answer these questions. Recently, several findings
have been reported such as, rhodopsin-like protein
in Chlamydomonas and Paramecium, chemotaxis
of Escherichia coli for methamphetamine and GA-
BA receptor in Escherichia coli. There is a close
Similarity in the mechanism of phototransduction and
olfactory transduction in vertebrates. Similarities
also exsist between neurotransduction in the higher
organisms and bacterial chemotaxis. These mechanisms
consist of three steps, signal reception, transduct-
ion of its information into the intracellular
machinery and response to the signal.
I would like to propose a hypothesis concerning
the origin of multi-cellular organisms. Higher
organisms, including our bodies, are composed of
many kinds of uni-cellular organisms some of which
showed phototaxis or chemotaxis. The longest period,
about three billion years, in the history of life
was dominated by the microorganisms. This period may
have been enough for the evolution of most of the
functions of the higher organisms today. The fusion
of the nucleus after that of the plasma membrane
resulting in a hybrid cell may be a remnant function
of the early evolutionary period.
PHYLOGENETIC POSITION OF PSEUDOCOELOMATE
PHYLA INFERRED FROM NUCLEOTIDE SEQUENCES OF
18S RIBOSOMAL DNA.
R. Ueshima!, T. Kobayashi”, Y. Shirayama? and N. Satoh?.
Mnstitute of Biol. Sci., Univ. of Tsukuba, Tsukuba, 7Oceanic
Res. Inst., Univ. of Tokyo, Tokyo, *Dept. of Zool., Fac. of Sci.,
Kyoto Univ., Kyoto.
Phylogenetic position of pseudocoelomate phyla,
especially the phylogenetic position of Priapulida and
Entoprocta, has long been controversial. In order to
understand the molecular evolutionary relationships, we
amplified partial 18s ribosomal DNA from five
pseudocoelomate phyla, Rotifera, Priapulida,
Nematomorpha, Acanthocephala and Entoprocta, and two
acoelamate phyla, Ctenophora and Nemertea. Nucleotide
sequences for approximately 1.0 Kbp were determined by
direct sequencing and were compared with those of other
animal phyla. Dendrogram constructed by neighbor-
joining method with Cnidaria and Ctenophora as outgroups
did not support the monophylety of pseudocoelomates and
the closer relationships between Entoprocta and
Tentaculata. Systematic position of each pseudocoelomate
phylum was however ambiguous, due to the accelerated
mutation rates and frequent insertion or deletion events in
pseudocoelomates. Forthcoming sequence data on
remaining regions of 18s rDNA and exclusion of
hypervariable sequences will help to elucidate the correct
relationships.
BIOCHEMICAL SYSTEMATICS OF THE FIVE STARFISH
SPECIES OF THE FAMILY ASTERIIDAE FROM JAPANESE
WATERS.
N. Matsuoka, K. Yoshida, K. Fukuda, M. Sugawara,
and M. Inamori
Dept. of Biol., Fac. of Sci., Hirosaki Univ.,
Hirosaki.
The phylogenetic and evolutionary relationships
among five starfish species of the family
Asteriidae of the order Forcipulata from Japanese
waters were studied by enzyme electrophoresis.
The five species examined were Asterias amurensis,
Aphelasterias japonica, Distolasterias nippon,
Coscinasterias acutispina and Plazaster borealis.
From the allozyme variation observed in 15
different enzymes, the genetic distances between
species were calculated and the biochemical
dendrogram for the five species was constructed.
The biochemical dendrogram showed the following:
(1) The five species are divided phylogenetically
into three groups: 4. amurensis and P, borealis
A. japonica and D. nippon, and C. acutispina.
(2) A. amurensis and P. borealis is the most
closely related to each other. (3) A. Japonica is
more closely related to D. nippon than to other
species. (4) C. acutispina is the most distant
species of the five members. The electrophoretic
results are well consistent with the immunological
study using enzyme inhibition test, although they
do not support the Fisher’s taxonomic system based
on the morphological studies. The evolutionary
process of the five species was discussed from the
present biochemical dendrogram and non-molecular
evidence.
Taxonomy and Systematics 1301
PHYLOGENETIC ANALYSIS OF DEUTEROSTOMES ; THE
ANCESTORS OF VERTEBRATES AS DEDUCED FROM
NUCLEOTIDE SEQUENCES OF rDNA .
H. Wada’ , M.Nakauchi*, N.Satoh’.’Dept. of Zool. , Fac. of
Sci. , Kyoto univ. , Kyoto . * Kochi univ. , Kochi.
The question "how vertebrates evolved ?" has long been
debated. Some pieces of evidence from molecular phylogeny
support the popular Garstang's theory. We have studied this
question. 18S rDNA sequences of echinoderms
(Strongylocentrotus intermedius , Asterias amurensis) ,
hemichordate (Balanoglossus carnosus ), cephalochordate
(Branchiostoma caribaeum), urochordates (Ciona intestinalis ,
Halocynthia roretzi, Thalia democratica , Oikopleura sp.) were
obtained by PCR-direct sequencing method using 4 -
exonuclease. Phylogenetic trees were constructed by neighbor-
joining method ,maximum likelihood method and maximum
parsimony method. All of these trees show that echinoderms and
hemichordate have a common ancestor not shared by other
deuterostomes. We could not determine clearly the evolutionary
sequences among this group; echinoderms and hemichordate ,
cephalochordate ,urochordates and vertebrates, except that
urochordates and vertebrates are a little closely related to each
other But the results suggest us a very interesting information;
these deuterostome groups had not evolved sequentially , but
diverged in a very short time. We are now confirming this by
28S rDNA sequences.
ALL OF THE CONSTITUENT CHAINS OF THE HEMO-
GLOBINS AND MYOGLOBINS ARE NOT HOMOLOGOUS
T.Suzuki
Dep. of Biol., Fac. of Sci., Kochi Univ. Kochi
Hemoglobins and myoglobins are some of the
best studied proteins. They are distributed in
animals, plants and bacteria, and the characte-
ristic two intron-three exon structure is widely
conserved in animal globin genes. To date, all of
the hemoglobins and myoglobins are believed to
have a common origin, and so they are considered
to be homologous. We have isolated a completely
new type of myoglobin from the red muscle of the
abalone Sulculus diversicolor aquatilis. The myo-
globin consists of an unusual 41 kDa polypeptide
chain, contains one heme per chain and forms a
homodimer under physiological conditions. The
cDNA-derived amino acid sequence of Sulculus myo-
globin showed no significant homology with any
other globins, but, surprisingly, showed high
homology (35% identity) with human indoleamine
2,3-dioxygenase, a tryptophan degrading enzyme
containing heme. This clearly indicates that Sul-
culus myoglobin evolved from a gene for indole-
amine dioxygenase, but not from a globin gene
(Suzuki & Takagi (1992) J.Mol.Biol. in press). In
addition, we recently found that a part of the
linker chains of annelid giant 4,000 kDa hemo-
globin is homologous with the ligand binding
domain of vertebrate LDL-receptor (Suzuki & Riggs,
unpublished result). These data indicate that all
of the hemoglobins and myoglobins are not homolo-
gous, and suggest a pliable molecular evolution.
MOLECULAR PHYLOGENY AMONG THE PHYLA POGONO-
PHORA, VESTIMENTIFERA AND ANNELIDA.
AN APPROACH FROM HAEMOGLOBIN SEQUENCES
H.J.Yuasa, T.Furukohri, T.Suzuki, T.Takagix,
N.Suzuki*+. Dept. of Biol., Fac. of Sci., Kochi
Univ. *Inst., Biol., Fac. of Sci., Tohoku Univ.
**Noto Marine Lab., Kanazawa Univ.
Oligobrachia masikoi, belonging to the Phylum
Pogonophora, contains a giant extracellular
haemoglobin with M.W. about 400kDa. This haemo-
globin consists of four haem-containing chains
(Al, A2, Bl & B2) just as in annelid and _ vesti-
mentiferan haemoglobin. Three of the four chains
were isolated by reverse-phase chromatography
and the N-terminal 30 residues of the chains
were determined directly by an automated
protein sequencer (Applied BioSystems 477A). The
remaining chain(Bl) was isolated by SDS-PAGE and
sequenced. The cDNAs for chains A1,A2,and B2
were amplified by PCR method using two primers,
an oligo-dT,~ adopter and a redundant oligomer
(23mer) sniesines on the basis of each N-
terminal amino acid sequence. The cDNA-derived
amino acid sequences of Oligobrachia haemoglobin
chains show high homology with those of Tylo-
rrhynchus (Polychaete, Annelida), Lumbricus
(Oligochaete, Annelida) and Lamellibrachia
(Vestimentifera). An evolutionary tree for these
haemoglobins was constructed. The tree strongly
suggests that Pogonophora, Annelida and Vesti-
mentifera evolved from a common ancestor, and
that Pogonophora is especially related with
Vestimentifera.
DIVERSITY AND EVOLUTION OF HEMOGLOBINS OF
THE ARCID CLAMS BARBATIA VIRESCENS AND
BARBATIA LIMA
T.Arita, A.Nakamura, T.Furukohri and T.Suzuki
Dept. of Biol., Fac. of Sci., Kochi Univ. Kochi
The arcid clam Barbatia virescens has hetero-
dimeric hemoglobin (HbI and II) in erythrocytes.
On the other hand, the congeneric clam B.
lima contains heterotetrameric hemoglobin and
polymeric hemoglobin consisting of unusual two-
domain chain. The purpose of this study is to
make clear the diversity and the molecular evo-
lution of bivalvia Hbs. The cDNAs encoding
these Hbs were first amplified by PCR method,
and then the cDNA-derived amino acid sequences
of B. virescens HbI (156 residues) and II (157
residues) and B. lima two-domain Hb (308 resi-
dues) have been determined. These Hb sequ-
ences were compared with those of other bibalves
Anadara, Scapharca, Lucina, Calyptogena and
Barbatia reeveana. An evolutionary tree for
these Hb chains was constructed. The branching
pattern of the tree essentially agreed with that
of the tree constructed with fossil record. The
tree suggests that the divergence of Barbatia
chain and Anadara chain has occurred after the
divergence of genus, and that the advent of
two-domain Hb chain has occurred before the
divergence of B. lima and B. reeveana. We sug-
gest that the arcid hemoglobin is relatively
less important for survival, so it evolved
rapidly and resulted remarkably diversity in
chain compositions and subunit structure.
1302 Taxonomy and Systematics
THE PHYLOGENETIC RELATIONSHIPS OF FREE
LIVING PLATYHELMINTHS BASED ON PARTIAL
NUCLEOTIDE SEQUENCES OF 18S rDNA
T. Katayama!, H. Wada’, N. Satoh’ and M. Yamamoto!.
1Ushimado Marine Lab., Okayama Univ., Okayama.
Dept. of Zool., Fac. of Sci., Kyoto Univ., Kyoto.
Platyhelminths are most primitive animals within the
Bilateria. Primitive Platyhelminths, acoel turbellarians, are
key animals to understand the origin and evolution of the
metazoa. We determined and compared nucleotide
sequences of about 1000-bp long central part of 18S 1DNA
of nine turbellarians, including one species of the group
Acoela (Convoluta naikaiensis ), six of the group
Polycladida ( Notoplana koreana, Planocera mutltitentaculata,
Stylochus orientaris, Pseudostylochus obscurus,
Stylochoplana pusilla, and Thysanozoon brocchii ), and two
of the group Tricladida (Dendrocoelopsis lactea and Dugesia
japonica). The deduced phylogenetic tree suggests an early
emergence of the acoel turbellarian Convoluta from the other
planarians. Complete nucleotide sequences of 18S rRNA
(or DNA) have been reported in several organisms. In order
to infer the phylogenetic position of the turbellarians, we
compared their sequences with those of other organisms.
The result supported the phylogenetic position of the acoel
turbellarian. Therefore, Convoluta may not be a case of
secondary reduction of higher acoclomates but one of the
creatures nearest to the metazoan ancestors.
MOLECULAR PHYLOGENY OF JAPANESE
SIKA DEER (Cervus nippon)
H. B. Tamate! and T. Tsuchiya?. 1Dept. of
Biotechnology and 2Dept. of Basic Sciences,
Senshu University of Ishinomaki, Ishinomaki.
Mitochondrial-DNA(mtDNA) restriction
sites were analyzed for four subspecies of
Japanese Sika deer. MtDNA was isolated from
C. n. centralis and cloned into a plasmid
pUC118. Blood samples were collected from
anaesthetized or hunter-killed animals. Total
DNA was extracted from the samples, digested
with restriction enzymes and hybridized with
the clones to detect mtDNA fragments. Three
invariant sites (two SaclII and Hpal) conserved
among vertebrate classes were identified in all
of the subspecies examined, i.e. C. n. centralis
(Honshu), C. n. centralis (Tsushima), C. n.
yesoensis, and C, n. yakushimae.
Intraspecific variation was studied by
comparing mtDNA restriction-site maps of
each subspecies. C. n. centralis (Honshu)
shared most of the restriction sites with C. n.
yesoensis, but not with C. n. centralis
(Tsushima) and C. n. yakushimae. The result
suggests that previous taxonomic status of the
subspecies, which is based on comparative
morphometrics, would need reconsideration.
PHYLOGENY IN LUCIOLINAE AND FLASH PATTERN
H.Suzuki’, Y.Sato*, S.Fujiyama? and N.Ohba?
‘Olympus Optical Co.,Ltd.,Hachioji, #Shinshu Univ.
Matsumoto and #Yokosuka City Mus. , Yokosuka.
The communication systems of Japanese fireflies
in Luciolinae were classified into three types
according to mating behaviors as follows (Ohba,
1983). HP system: Female responses at the
constant timing with flash to the pulselating
flash of male. LC system: Male seeks female with
synchronous flash. LL system: Female and male
communicate to each other with their original
flash patterns. In order to clarify the origin
of flash communication systems of fireflies, we
estimated the phylogenetic relationships among
eight species in Luciolinae by electrophoretic
analysis of allozymes. As the results, four
species of two genera(Hotaria parvula, UH.
tsushimana, Luciola yayeyamana and L. kuroiwae)
clustered one group and that corresponded to HP
system. Three species of two genera(L.
lateralis, Curtos costipennis and C. okinawana)
clustered one group and that to LL system. L.
cruciata made one own group and that to LC system.
The classification obtained here by biochemical
technique completely concordanted with the types
of flash communication systems even if each group
was consisted of members of different genera.
REPRODUCTIVE CAPACITY OF ALLOTRIPLOIDS
BETWEEN RANA JAPONICA FROM HIROSHIMA OR
ICHINOSEKI AND R. TSUSHIMENSIS.
M. Sumida and M. Nishioka. Lab. for
Amphibian Biol., Fac. of Sci., Hiroshima
Univ., Higashihiroshima.
Allotriploid frogs were produced from
crossings between R. japonica from Hiroshima
or Ichinoseki and R. tsushimensis by heat-
shock treatment of the fertilized eggs.
Almost all the allotriploids developed
normally and became male. Diploid hybrids
died at the larval or froglet stage. A
total of 65 male allotriploids including
reciprocal allotriploids between R. japonica
from Hiroshima and R. tsushimensis and
allotriploids between female R. japonica
from Ichinoseki and male R. tsushimensis
were completely sterile. On the other hand,
16 of 44 male allotriploids between female
R. tsushimensis and male R. japonica from
Ichinoseki were fertile and their spermato-
genesis was completely or partially normal.
The other 28 male allotriploids were
sterile. A total of 419 offspring were
produced from seven of the above 16 male
allotriploids by backcrossing with female
R. tsushimensis. They were all diploids and
of the R. tsushimensis type in external
characters. Most of them (96.9%) were
females. They showed the same electrophore-
tic patterns as R. tsushimensis at 14 loci
for enzymes and blood proteins. These
findings seem to indicate that some of the
male allotriploids reproduce by hybrido-
genesis, in which the R. japonica genome is
eliminated during spermatogenesis.
Taxonomy and Systematics 1303
CONTENTS OF CALCIUM AND _ INORGANIC
PHOSPHATE IN THE SKIN OF AMPHIBIANS
K. Uchida, Y. Ohtani, Y. Sasayama,
C. Oguro and H. Yosizawa!
Dept. of Biol., Fac. of Sei., Toyama
Univ., Toyama 930, and ‘Oral Anat. I,
Matsumoto Dental Col., Nagano 399-07,
Japan
It has been reported that in the skin
of some anurans, a large quantity of cal-
cium (Ca) is contained, especially in the
dorsal skin. However, it is not clear whe-
ther this phenomenon is general to am-
phibians or not. In the present study, Ca
contents in the skin of each five species
of anurans and urodeles were studied. In
addition, a species of Apoda which is an-
other group of amphibians was also exam-
ined as a reference. Furthermore, in-
Organic phosphate (Pi) contents in the
skin were measured, since Pi is also one
of the important minerals in hard tissues.
As a result, it was known that in every
species examined, Ca content in the skin
is the highest among soft tissues. Ca con-
tents in anurans are higher than those in
urodeles and in apoda. In completely
aquatic species of anurans, however, Ca
contents in the skin are relatively lower.
For Pi contents, the skin is not different
from other soft tissues in every species.
Taking these results into consideration,
it seems to be certain that anurans have a
larger quantity of Ca in the skin than
urodeles. The present results are dis-
cussed from a historical viewpoint of the
environmental adaptation of these three
groups of amphibians.
GEOGRAPHIC VARIATION OF PYURA MICROCOSMUS
(SAVIGNY) INHABITING EUROPEAN WATERS AND
THE RED SEA (UROCHORDATA, ASCIDIACEA)
T. Nishikawa’ and X. Turon? ‘Graduate School
of Human Informatics, Nagoya Univ., Nagoya,
Japan and *Facultat de Biologia, Univ. Bar-
celona, Barcelona, Spain.
Pyura microcosmus, commonly found in
European Warm temperate waters, has
recently been found to vary in the number
of right gonads in the Atlantic, while in
the Mediterranean there seem to be two
forms in terms of this number, segregated
bathymetrically. To study the extent of
this variation, we examined specimens from
the Red Sea, the Adriatic Sea, Bay of
Naples, NE Spain, Roscoff, Manche, English
Channel and Ireland, and also descriptions
from the literature. Detailed comparison
was made among these materials and the
other 3 European congeners. We conclude
that the gonadal variation should be
regarded as intraspecific, P. pantex re-
corded from the Red Sea being a junior
synonym of P. microcosmus. We propose a
taxonomic division of this species, in
terms of the percentage of individuals with
a single right gonad and the number of
gonadal capsules on each side, into three
forms: the Atlantic, the deep Mediter-
ranean, and the Red Sea / shallow Mediter-
ranean forms. Present distribution pattern
of the last form is not attributable to the
Lessepsian or Anti-lessepsian migration.
Rather, this form may have survived the
Messinian salinity crisis.
GENETIC VARIABILITY OF mtDNA AMONG THREE
TYPES IN Halocynthia roretzi -II-.
A.Hino!, T.Numakunai?, T.Kobayashi?, M.
Goto? , R.Ueshima?, K.Numachi?, 'Dept. of
Biol. Sciences, Kanagawa Univ. Hiratsuka,
@Mar.Biol.St., Fac. of Sci.,Tohoku Univ.
Asamushi,?Ocean Res. Inst.,Univ. of Tokyo.
Halocynthia roretzi is classified into
three types according to the difference
of the breeding season and the start time
of spawning. They seem reproductively
isolated and there is a possibility that
they represent an early stage of sympatric
speciation. To ascertain such possibili-
ties the restriction fragment polymorphism
of mtDNA was studied. The analysis of mt-
DNA restriction fragments was done as the
previous report. Nineteen individuals of
Type A, 18 of Type B and 21 of Type C which
were collected at Asamushi were tested.
The molecular weight based on agarose
gel electrophoresis of restricted mtDNA
was about 16kbp. No polymorphism with
few restriction site was found in the
digestion by Aval, BamHI, Bgll, BglII,
EcoRV, HindIII and PstI. In the case of
Xbal digestion, one adult of the Type C
showed unique pattern of the restriction
fragments. After HincII digestion, poly-
morphism was found and it was correlated
with each type. In Type B, no polymorphism
was found in all digestions which were
tested. All patterns of restriction frag-
ments of Type B were the same as the major
patterns of the Type A and C.
MOLECULAR PHYLOGENY OF ASCIDIANS BASED ON
NUCLEOTIDE SEQUENCES OF 18S rDNA AND 28S rDNA .
H. Wada’ , M.Nakauchi’, N.Satoh’.’Dept. of Zool. , Fac. of
Sci. , Kyoto univ. , Kyoto .” Kochi univ. , Kochi.
Ascidians has evolved various types of reproduction;colonial and
solitary. In spite of these life style, the orthodox taxonomic
classification categorized them into Enterogona and Pleurogona
based on the structures of gonad and some other characters.
Each group contains both life styles. In order to examine this
classification from molecular phylogeny, we determined the
sequences of the central regions of 18S rDNA by PCR-direct
sequencing method using A -exonuclease .By neighbor-joining
(NJ) method the phylogenetic tree was constructed from the
sequences of seven Enterogona ascidians ,nine Pleurogona
ascidians and human and Xenopus.
In this NJ tree nine Pleurogona ascidians and seven Enterogona
ascidians form discrete groups irrespective of their life styles.
Therefore the present molecular phylogenetic analysis supports
the classification that emphasize that the two life styles had been
acquired independently several times . As for the relationships
within the two orders the NJ tree is approximately consistent with
orthodox classification except for two Ciona species. Two Ciona
species, Ciona savignyi and Ciona intestinalis which are very
similar in their morphology do not form a single cluster
However the tree constructed from 28S rDNA of six Enterogona
ascidians supports the close relationship between the two Ciona
species. We concluded that two Ciona species are closely related
genetically, but the substitution rate of 18S is too slow to explore
the relationships within the orders in ascidians.
1304 Taxonomy and Systematics
Genetical valiability in rRNA gene inCiona.
M. Matsumoto, T. Shimada and M. Hoshi
Tokyo Inst. of Technol., Kanagawa
Genetical variability in rRNA gene (rDNA)
repeated units was studied in Ciona savignyi and
C. intestinalis. We had previously reported that
rDNA of Ciona has restriction fragment length
polymorphysm (RFLP). For further investigation,
genetical variability of spacer region of rDNA of
Ciona was examined by polymerase chain
reaction (PCR) method. C. savignyi were collected
in Kesennuma, Onagawa and Tateyama. C.
intestinalis were collected in Onagawa, Toyama,
Maizuru, Nagoya, Hiroshima and Kouchi. Nuclear
DNAs from the individuals were extracted. The
primers for PCR were 23 mer oligomers
containing 3' end of 18S rDNA and 5' end of 5.8S
rDNA , then the spacer region between 18S rDNA
and 5.8S rDNA were amplified. The amplified PCR
products were classified into 22 groups in C.
intestinalis and 10 groups in C. savignyi in length.
It was found that, these wide valiability in spacer
region of rDNA mainly depends upon collected
areas.
LARVAL STRUCTURE, POST-LARVAL DEVELOPMENT
AND EARLY ASTOGENY OF THREE CELLEPORINA
FROM HOKKAIDO, JAPAN.
H. Ikezawa and S. F. Mawatari. Zool. Inst.,
Fac. of Sci., Univ. of Hokkaido, Sapporo.
Larval structure, metamorphosis, ances-
trular form and budding pattern of three
Celleporina species (C. porosissima, C. sp.
1, C. sp. 2) from Hokkaido were studied by
scanning-electron and light microscopy. The
three species are basically alike in the
following three items: 1) Larvae have an
extensive corona, a relatively small palli-
al sinus and a developed metasomal sac, 2)
the cystid epidermis is derived from the
wall and roof regions of metasomal sac, 3)
ancestrulae are single and a type of celle-
porioid. In addition to some minor differ-
ences in their histology, an important
difference was seen between C. porosissima
and the other two in the shape of ancestru-
lae: The ancestrulae of C. porosissima have
a budding zone around the distal half from
which the first daughter zooid makes its
appearance; those of the other two species
have a pair of disto-lateral projections
that bud off a pair of first zooids simul-
taneously. A combination of the characters
in the ancestrular morphology and early
astogeny suggests C. sp. 1 relates more
closely to C. sp. 2 than to C. porosissima.
BIOSYSTEMATICS OF THE GENUS STYLACTARIA
(HYDRACTINIIDAE, HYDROZOA) FROM HOKKAIDO,
JAPAN.
H. Namikawac, Seed oe Mawatari2, | ept. of
Zool., Natn. Sci. Mus., Tokyo, “Zool.
Inst., Fac. of Sci, Hokkaido Univ.
Sapporo.
Mode of reproduction, morphology and
behavior of planulae, and substrate
specificity were studied and compared in
three species of the genus Stylactaria
from Hokkaido, S, conchicola, S, uchidai,
and S, multigranosi. Observed differences
in these attributes provide additional
characters for discrimination of the
three species. Stylactaria conchicola is
oviparous, its planulae are sedentary,
and its hydroid is substrate specific.
Stylactaria uchidai differs in being
ovoviviparous, in having crawling
planulae, and in being a substrate
generalist. Stylactaria multigranosi is
parthenogenetic, its planulae are
sedentary, and it is substrate specific.
Correlations were noted between
attributes of the planula larva and
substrate specificity in the three
species. The phylogenetic significance of
different gonophore types in the genus is
cosidered.
MORPHOLOGICAL OBSERVATIONS ON CERATA OF
AEOLID NUDIBRANCHS (2)
K.Okawa,Dept.of Biol.,College of Lib-.Arts,
Hirosaki Univ.,Hirosaki,Aomori.
Most species of aeolid Nudibranchs have
stored nematocysts (cleptocnidae) in
cnidosacs from coelenterate upon which they
feed. Phyllodesmium serratum feeds on
octocorallian coelenterate (Cornularia
Sagamiensis) and has a terminal sac
(Rudman,1981) at the tip of cerata, in
which no nematocysts are found. The terminal
sac of P. serratum and the cnidosac of
Hermissendg crassicornis and Cratena
lineatg were examined by light-and electron
microscopy. To ascertain structural
constitutions, the regenerated terminal sac
of P. serratum and the regenerated cnidosac
of H. crassicornis were also observed. The
observations suggest that the terminal sac
has a degenerated structure of the cnidosac
and it's condition is nil functional for a
storage of nematocysts. A close contact
between differentiating digestive gland
cells and messenchymal cells occurred
during morphogenesis of the regenerated
cnidosac, being accompanied by a cellular
proliferation from the distal part of the
digestive diverticulum, but a celluar
proliferation mostly from messenchymal
cells was occurred during regeneration of
the terminal sac.
Taxonomy and Systematics 1305
SCANNING ELECTRON MICROSCOPIC OBSERVATIONS ON
SPICULES, GEMMULE COATS, AND MICROPYLES OF THE
FRESHWATER SPONGES, EUNAPIUS SINENSIS (ANNANDALE)
Y. Masuda and K. Satoh, Dept. of Biol., Kawasaki
Med. Sch., Kurashiki.
In Japan, Eunapius sinensis was first collected
from Lake Kawaguchiko in Yamanashi Prefecture and
Lake Ashinoko in Kanagawa Prefecture by Sasaki in
1933. This species was collected from Hyakken
River in Okayama Prefecture in 1987, and is here
recorded for the third time in Japan.
Sponge was flat, forming spreading thin layers
without any projecting branches. Megascleres
were feebly curved, amphioxea, entirely smooth ;
length range 192 —- 295 um, width range
8.1 - 15.3 wo. Microscleres were absent.
Gemmoscleres were rather robust, usually slightly
curved amphioxea, ranging from smooth to covered
with few to many large and irregular spines ;
length range 37 - 110 wn, width range
4.8 - 8.7 ym. Malformations of gemmoscleres were
quite frequent.
Gemmule were somewhat depressed, subspherical,
forming pavement layer at base of sponge, firmly
adhering to support. Pneumatic layer were more
or less developed, of varing thickness, consisting
of polygonal prismatic alveoli and forming a
continuous coat over gemmules. But at the base of
gemmule adhering to support, pneumatic layer
lacked alveoli. Gemmoscleres were embbeded
tangentially in pneumatic layer in great
numbers. Micropyle was tubular. Porus tube was
slightly protruded from the surface of pneumatic
layer.
PYRGOPSELLA, A SPONGE-INHABITING "CORAL-
BARNACLE” (CIRRIPEDIA: PYRGOMATIDAE), WITH
REMARKS ON CIRRI.
M.J.Grygier. Seto Mar. Biol. Lab., Fac. of
Sci., Kyoto Univ., Shirahama, Wakayama.
Pyrgopsella Zullo is the only genus of
Ppyrgomatid barnacles associated with spon-
ges rather than corals. Only P. annandalei
(Gruvel) from the Andaman Islands and P.
stellula Rosell from the Sulu Archipelago
have been described, with one record each.
The late T. It6 collected sac-shaped
barnacles, identified here as P. stellula,
from a sponge in Tanabe Bay, Wakayama Pref.,
Honshu. This extends the species' range
from the Philippines to Japan. An undes-
cribed species occurs in a sponge from nor-
thern Sulawesi, Indonesia, and differs from
P. stellula mainly by having a relatively
smaller shell wall, much narrower and
smoother opercular plates, differently
shaped external cuticular spines, and tri-
angular distal spines on the coxa of cirrus
IV. The use of positional rather than
morphological terms to distinguish the two
rami of each ‘cirrus (thoracopod) is usual
in descriptions of barnacles. In Pyrgop-
sella serially homologous rami — as demon-
strated by the musculature — functionally
serve as either the outer or inner ramus on
different cirri. To account for this, a new
morphological interpretation of the rami is
tentatively proposed.
This work was supported by a JSPS Post-—
doctoral Fellowship awarded through the NSF
Japan Program.
The first description of laboratory-reared larva of
pomatochelid crab, Pomatocheles jeffreysii Miers,
1879 (Crustacea: Decapoda: Anomura).
K. Konishi! and M. Imafuku?- 'Natn. Res. Inst. Aquaculture,
Mie and 2Dept. of Zool., Fac. of Sci., Kyoto Univ., Kyoto.
The larvae of most primitive hermit crabs of the family
Pomatochelidae have been very poorly described, in spite of its
significance in the systematics of the infraorder Anomura.
Forest (1987) described two juvenile specimens of
Pomatocheles stridulans Forest, but no larval studies which
based on laboratory-reared materials have been represented to
date. We collected an ovigerous female of Pomatocheles
jeffreysii Miers from off Anori, Mie Prefecture, 100m in
depth, and obtained the first stage larva under laboratory
conditions. The female carried only 5 eggs of 1.43 XK 1.11mm
in mean length. The newly-hatched larva covered with very
thin cuticle, showing prezoeal features. Antennal exopod
which can be found through the prezoeal cuticle is reduced,
while pereiopods and pleopods are well developed.
Furthermore telson bears 13 pairs of posterior processes, and
no anomuran hair is found among them. These features
indicate that the larval development of P. jeffreysii is a typical
abbreviated form.
SYSTEMATIC AND BIOGEOGRAPHIC REVIEW OF
PINNOTHERID CRABS FROM JAPANESE WATERS
M. Takeda’ and K. Konishi? !Natn. Sci. Mus., Tokyo and
2Natn. Res. Inst. Aquaculture, Mie.
Crabs of the family Pinnotheridae are mostly known as
commensals living in mantle cavities of bivalve shells, while
some peculiar species are associated with other invertebrates,
e.g., gastropods, sea urchins, holothurians, and ascidians.
Free-living species are also found in soft muddy bottom.
According to Schmitt et al. (1973) and some recent papers, ca.
280 species are described in this family which consists of five
subfamilies: Pinnotherinae, Pinnotherelinae, Asthenognathinae,
Xenophthalminae and Anomalifrontinae. Altogether 30 species
of 13 genera have been recorded from Japanese waters. viz. 9
spp. of Pinnotheres, 2 spp. of Pinnaxodes, | sp. of
Durckheimia, | sp. of Orthotheres. | sp. of Xanthasia, 1 sp. of
Ostracotheres and 4 spp. of Sakaina (Pinnotherinae); 5 spp. of
Pinnixa, 1 sp. of Pseudopinnixa and 1 sp. of Terrias
(Pinnotherelinae); 1 sp. of Asthenognathus and 2 spp. of
Tritodynamia (Asthenognathinae); 1 sp. of Xenophthalmus
(Xenophthalinae). We examined some specimens identified
with additional 3 species: Pinnotheres sp. from Amami-Oshima,
Ostracotheres sp. from Okinawa and Pinnixa sp. from the coast
of Iwate Prefecture. Fifteen species are endemic to Japanese
waters and most of the others are restricted to the East Asia.
Their distribution is dependent upon that of the hosts. The free-
living species are not always found commonly in the whole
distnbutional ranges probably due to their special habitat.
1306 Taxonomy and Systematics
TAXONOMICAL AND ECOLOGICAL ASPECTS OF
3 NEW SPECIES OF POTAMALPHEOPS POWELL
(ALPHEIDAE, DECAPODA, CRUSTACEA)
Y. Miya. Lib.Arts, NagasakiUniv. Nagasaki
Illustrated description of 3 new species
of Potamalpheops are provided. First Spe-
cies from Pecehmater zone of SungeiSelange
in Malaysia, the second from mangal flats
in Sungei Buloh, Singapore and in Adelaide
River, NT, Australia, and the third from
freshwater zone in Calabar River, Nigeria.
Malaysian species is also of special in-
terest on account of its specialized adap-
tations to carrying large (0.96x0.68 mm at
pre-eyed stage) few (3-15) eggs. Among 41
mature females (cl 2.90-4.50 mm) an unusu-
al lamellar expansion of Ist and 2nd pleu-
ra shows increasing 14Z and 11%, respec-
tively, in height of somite. Eggs attached
only to lst-3rd pleopods are enclosed ven-
trally as well as laterally by the expan-
sion. Another specialized structure of
carrying eggs is the same lamellae of lst-
4th pleopods as found in P. monodi(Sollaud)
from West Africa. An examination of whole
eggs just before hatching (12 eggs, 1.14 x
0.79 mm) carried by the female (cl 4.25mm)
suggests the species hatching as a highly
advanced zoea. It has movable eyes, lst
maxilliped-lst pereopod biramous, 2nd, 3rd
and 5th pereopods uniramous, 4th absent,
no pleopod buds, tailfan with 7+/7 setae,
and uropod buds undifferentiated. A key is
also provided to the 8 species of Potam-
alpheops Powell, 1979.
AXIIDAE COLLECTIONS MADE BY DR. TH.
MORTENSEN'S EXPEDITION TO JAPAN
(CRUSTACEA, THALASSINIDEA) .
K. Sakai, Biological Laboratory, Shikoku
University, Tokushima.
The collections of the family Axiidae
in the Zoological Museum, Copenhagen,
were examined. They were made during Dr.
Th. Mortensen's Expeditions in the Indo-
Pacific region from 1899-1930 (the
Pacific Expedition in 1913-16, the Danish
Expedition to the Kei Islands in 1922, and
the Java-S. Africa Expedition in 1929-30)
and the Galathea Expedition in 1950-52.
As a result, the occurrence of 14 spp.
containing 5 spp, 7 new spp, and 2
uncertain spp. are documented. In
Mortensen's collection 2 new Japanese spp.
Eiconaxius mortenseni Sakai, 1992 from
Sagami Bay, Axiopsis tsushimaensis Sakai,
1992 from Fukuoka, Korean Strait, and
another species, Eiconaxius farreae from
Sagami Bay, are recorded, although A.
tsushimaensis was defined before as an
affinity of A. serratifrons sensu Sakai,
1970. (Zoologica Scripta, 21(2), 1992)
1307
ANNOUNCEMENTS
THE 64TH ANNUAL MEETING OF THE ZOOLOGICAL
SOCIETY OF JAPAN
The 64th Annual Meeting of the Zoological Society of Japan will be held at Okinawa from
November 20 to 23, 1993. Further information and application forms will be sent to the
domestic members in the ‘Biological Science News’ (No. 1). The deadline for application is
August 16, 1993.
For application from abroad, please contact:
Professor K. Yamasato
Organizing Committee of the 64th Annual Meeting
Department of Biology, College of Science
Univesity of the Ryukyus
Senbaru 1, Nishihara
Okinawa 903-01, Japan
Phone: 098-895-2221
ZOOLOGICAL SCIENCE AWARD
Annual awards for the best original papers have been established through the donation of
Narishige Scientific Instrument Laboratory, Tokyo. The sum of about 500,000 yen will be
awarded annually at the Annual Meeting of the Zoological Society of Japan to a few papers
published in ZOOLOGICAL SCIENCE during the preceding calendar year. Every original
paper published in this journal will automatically be candidates for the award. The aim of the
award is to encourage contributions to this journal. Selection Committee for the award will be
organized every year.
ZOOLOGICAL SCIENCE AWARD 1992 was given to the following four papers.
Matsuoka, N. and T. Hatanaka: Molecular evidence for the existence of four sibling species
within the sea-urchin, Echinometra mathaei in Japanese waters and their evolutionary
relationships. Vol. 8, No. 1: 121-133 (1991)
Fujimura, K., F. Yokohari and H. Tateda: Classification of antennal olfactory receptors of
the cockroach, Periplaneta americana L. Vol. 8, No. 2: 243-255 (1991)
Takahashi, S. and M. Miyatake: Immuno-electron microscopical study of prolactin cells in
the rat: postnatal development and effects of estrogen and bromocryptine. Vol. 8, No. 3:
549-559 (1991)
Wilder, M. N., T. Okumura and K. Aida: Accumulation of ovarian ecdysteroids in
synchronization with gonadal development in the giant freshwater prawn, Macrobran-
chium rosenbergii. Vol. 8, No. 5: 919-927 (1991)
1308
ACKNOWLEDGMENTS
The Zoological Society of Japan and the Editorial Committee of Zoological Science are
grateful to Narishige Scientific Instrument Laboratory Co., Ltd. (President Mr. Eiichi Narishige)
for the financial aids for publication of Zoological Science, Volume 9 and constant support for
Zoological Science Awards for 1985-1992.
1309
AUTHOR INDEX
A
Aida, Katsumi ........... 37, 375, 607, 765, 1047
ENTITY OKO eee scl Fae own a Slane etla nee 305
ANI NYY AGT TCE ct ee oe Ae ee 349
Amano, Masafumi ..................... 375, 765
PNIMANO MW MUNOTU: .. ccc ss wees sa aes odenmiote 749
Amemiya, Shonan .....................00005 897
PANG OMROICHI Gs. 52s ced cans nae ovaed ness bees 823
PNA SUSCIMEH se os ch shs soins ates aac ae 823
PMVAI Pe WASUMMASA, occ ccc naa oss as okihees oiees 223
ANTEN ENE) 21000 I eee eA 1061
Arane daly Silvial fcc. soascse se. eee tieetls 719
AsadassNobulhiko) -445... 0525 -aee enters 397
Asahinia, Kayoshi ......2:2.2.+...- dese sees 607
PXSATINIEPINOWIGIM «5.0! ceca spans aac wo SODA Baa euelns 1001
PAITIVAR NATSU sd oc. ccs va a oad wean ardh 47, 941
/NTAUTINES IM EST ee eA 941
B
Ba ding@Nastil® gains: cos eens sce cn tool Musee 1037
Berniwdowanrd Ac .6<jocs.05. ERS. AR: 7715
IBOckMaWiallten js.) sj geen gece nc eel 697
IB OULEMIVABCHION oi Lon sc teva vee ee AEE Aen hbk 639
Boxshall, Geoffrey A. ................. 859, 875
C
Canguilhem, Bernard ....................... 719
GIS WARS It ois. ssas le nieeoleiece go DE 227
Chiba sVoshihiko) < occ ccc ees: . Ode See 185
(ClARGEY, Gis croc Hen a erent caren Re bee 1025
Coland Claude)... cticusse soscawecceegatcc be 533
Coo PerWE ebay. asec cee detaane Ha seee suede wes 551
Crawford, Michael A. .....................00. 315
D
D’Antonio, Mauro .......................... 365
de Jesus, Evelyn Grace ..................... 633
de Linares, Liliana Vergara ................. 671
de Santos, Silvia Valentinuzzi ............... 671
DGhIOne Mig Misys: a coca. Ree 1025
DohkewKenyinOiien se ocnsecsccss sae eeere ae 119
E
EndomkKatswhikOpiage cscs eccuce «atest ane 725
F
Babrya leather f...2inicaece anes antes eet ee 995
ASAN OMS IW see once sleie eobeocre tine ore ets 365
Bs MOMOK ON oecsieserec wes ecaclatv sco Ae 329
UW INOMYWKIO) seve cc enone cteweneeonenees 329
Fujisaki AiO’ oiccs ceca cn aoc tee teds sees 741
RujiwartascNKiKO! 4.0. ickagancicn dies ceca mes 329
Fujiwara, Kyoko ..............0.e cee ee eee eee 397
Fuyjiwaras Shigeki .22..-.--.2.0--+-ke aaa: 569
Rukudas Masantieeee ceca oceeee seen te: 223
KW YW: sess ates ee soretls er SNE Des 445
Kukuta tShOpiG canes csnnieqedcnacacs ta eelenas 947
Funakoshi, Kimitake ........................ 445
Bunakoshiy Syojii®:-.s4...+2.-025+505.naeee 541
Furukawa, Kiyoshi .................... 607, 1047
Bunuyas bidetake 2 ..assc0500-ssanaeenereee 423
G
Georges ohms, 22) oo coc oes nano Me 675
Gerencser, Geroge A. ..............-2.0-05- 925
Ghebremeskel, Keb ......................... 315
GobbettisAMnaec:...5. .casc.08e SA 639
Gorbman, Aubrey ......................005 387
GoOtosMar Chin xs asics crates ccs VAR ee 211
Griffond, Bernadette ....................... 533
H
Flame Chan Pelee sae e mcs cs ssh) Peete! 37
Hanaokas Youichil s4.-555.6.-o9eseeens.s oe 175
lamyW Sa One see y- ce cks eas cence 37, 1047
UAT AR BNC IK Ones e sacs cyoiere. fo ciaroie nian Se sie) ste 211
lala, WOMANS coodccooceocscocdonsoaanbeuCe 211
RlanbigeMbaunenCeiSs...4.5.- 92> eee ee 5 ae 315
Harmonie Mamie Sis occ cane bo aioe wdoeek 275
Hasegawa, Yoshihisa ................... 375, 765
1310
Hashimoto, Ryuhei .......................5. 223
asumin Masatomernene ee ereee eee eee CREE rer 1093
Hayashiiensichi@nssa- pane ete eeere at 199
ISENO, WETS 5c ccoscoccncsncec0c0ocsteK 193
Hedricks Jerny e358. 2a a eee 995
Hihara,, Fuywore.cecaare nce eee eas 397
lSHieIO), WEVA coccogsvodcanodscn0000 633, 1061
Mirata: Junk@n. feccedclehorcee ceric ee eeeece 207
Prose JE WICH. nce cies crn ee oe 405
Bushida.VOmico: j22c5255-ecke eee 1087
Horita: PAKUSMI 2.5 .eccuus oieric eee 413
Horiuchi Shintt- 22-7265 eee eee 211
Muang ZN sos ch vena d che nee oe eae 113
ERUYS: JROMY. osseaicted nocjeomaennaeeeeer 859, 875
Ely odo; SUSUMU js)... 52 o ese eee eer 157
I
Ichikawa, Tomoyuki ........................ 305
ligo, Masayuki «acc sates. scenes 1047
ino, WenwhikoO. . ssc ec eee 119, 337
Tkdta; Takaaki rias.okcccnccuatiacnne ty soap aee 947
Ikeda, AinOsht js ssectscn oan seen ie ane 397
Inoue, Chiemit..«.5..c..0¢00.5.0 ee eee 757
Till; WaSWO | <a sescsseGcorvste overs aw aves Mra eee 633
IpY Ueno: cco3insa connie same eons 227
Ishii; Susumesisscccdencds eee 1017
ISMN, SAME A, s25cccc0000000c0G000000000 601
IshijimasWakashieeeperee er eer eee eer errr 785
Ishikawa, Yuli -.o5cc0 Gene waraneatineen eee 1067
[tos Tatsunony ...c.355.ac ee os eee 859, 875
MUCHA; WChirO) «weg sccdccnharna adh RR Eee 891
Iwamatsu, Takashi .....................0000- 589
Iwasaki, Naohiko ...................0055 47, 941
IwasawacAtsushy «otc... See Roe eee 175
Iwasawa, Hisaaki ..................... 439, 1093
J
Jacob Mantamiman ener eee eerie 457
John, Mathew, Ty.c.c:5,3:<0.0060 sae ee eee 675
K
Kasotant) RYOKO ironies oe osc sicvee 3 eeeetane oe: 741
Kai-va Imo y uk... 3 ines mete telemtae teeta seetane 785
Kakinumias sOSHIKO Nene ce creneeeniererciieile 757
Roa os YOSHIKI oo o.c 5: ccacsce!sss crete eee = fer 725
Kamba, Mari. ..0.02..000000.+ 0s cee 169
Kanatani, Kazushi ........................- 1009
Kaneko; TOyOji) .....606 cece te 1061
Kanzaki, Ryohei=-23)---4eeeeeeee eee 241, 515
Kasuya® Biiti) <i) 4.020200. es eee Pee 469
Kataoka; Hiroshi’... 2.3’. 0). eee 149
Katayama; Isaovs?..2!........424.ce ee eeeeee 337
Kawashima, Seiichiro .... 293, 343, 357, 649, 791
Kearn, Graham C.. ....... 0...0.20gses eee 451
Kezuka;, Hiroaki ...........<0...0.8Geee eee 1047
Kihara, Hirozi K. ..............0.. +) aeeee eee 337
Kikuta, Toshiteru ............: eee eee 463
Kim, J. Gs oes. cose ad bs o.0000 oe eee 583
Kimura, Fukuko..<......:.:..0.0:see eee 223
Kimura, Masami --......... 4.2 eee 1009
Kimura, Tetsuya ....... 2+. s.c2 ee eee eee 58
Kirtishi, Shonosuke .......................-- 149
Kishida, Yoshikazu ......................-.- 575
Kishigami, Akio ...........:: .:4se eee 211
Kobayashi, Hideshi ......................... 619
Kobayashi, Hirokuni ....................... 1087
Kobayashi, Makito ...................-.-.0- 765
Kobayashi, Yuta ....... 22.2554: 3-0 eee eee 791
Kojima, Manabu) Ke. 4. 5-e- eee eee 741
Kosaka, Toshikazu 5---5.- 1. eee ene 101
Koshidas Yutakalss) eee 211, 423
Kubokawa; Kaoru ©.....-.. 40.2 oeeee eee 1017
Kubota; Shini...2% «06.5 doo. eee 231, 413
Kumaki, Toru) ..... 03 s.0s.0...00s00 eee 469
Kurabuchi, Shingo .......................0-. S15
Kuroda, Yukiaki «2... :....+s.06 eee eee 283
L
Eawrence, John M: 2522.4... 0... eee 321
Lee, Chang Eon ............. +: 00 971
Lee} Young Hwan .............sasnseeeeee 971
Lim, TM... ..o.0s0s00:.c0000 sos 20 eee 665
M
Machida, Takeo) oc. .000 0... one eee 357
Madsen StetfeniSs .ac0s--.- 06 -ee eee eee TiS
Maeno, Yukio... 0.5 cco ncec 0s tcteeidd ener 451
Makabe, Kazuhiro W. .................-005. 569
Mansour, Mohamed H. .................... 1037
Maruyama, Koscak .................00eeeees 219
Matsufuji, Mitsunori .....................08- 725
Matsui, Masafumi ....................0.00005 193
Matsumoto, Akira .................. cece eens 679
Matsumoto, Wadao ......2....2.ccc0ssecesnee 835
Matsuoka, Tatsuomi ......................5- 529
Matteo, Loredana Di ....................... 365
Michibata, Hitoshi ...................0..0005 207
IMDM CCHS ETEIO) oo ee cess esc receteiw nceieve je winnie se ated 365
NOMIC ATINIBUC ete o clecmg yas auras wiseteeee ef eee 719
IMiitasIViaSatOShi .......005 cca scence cea eneulens 563
INITUTARILWaAOs cack nkanada a dhe ees LRN 947
MiiyataeShOhell esnnscccesras see h oes een 337
IMOhmi Ide OWL es cx .ccad dace dees ee 601
MohnibloshikO\...25.0.2.0.205 04 60) ee ek 601
IMOranRetena kick: iecc nash ia & etna eee 321
IMOnmMnAKAO) 2 nececc ccc cascade ee 343
Montabliromicht ..........:h-see-sesstee nen 1
Murakami, Masahiro ........................ 891
N
INapaireVukaiUmil.:. 622240 secaceten te seek sete 475
Nagaishi, Hiroshi ....................0.-0000- 65
Nagasawa, Hiromichi .....................5. 149
Nagatomi, Akira ...............0.0e0e eee eee 843
INaitoh TOmiO: <i oncccscccd elle eee ee! 713
Nakagome, Yasuo .......................-.: 601
Nakajima, Katsuyuki ...................... 1009
Nakajima Yoko's tenccecccec ede eet ecb lee 897
Nakamura, Shogo ....................0eee eee 741
INakauchil, YUNI case. dccccaveseetteswcteseees 219
Nakaya, Kazuhiro ..................0.0e eee 811
Nakazawa, Toshiyuki S...................... 357
INamiuka gEide®: 22: accsscisisesarod eee eutese 463
Nishibe sYVasuo® eacc06saccccacdee escent 337
INiShidastainOki: csec6ccccceesscaneede aes veces 569
Nishikawa, Teruaki ....................00005 405
IN@MIUTARME ICID, -oie:e 5s s:oeus erste ores ovoselosars Gioesereva, 199
INOUMUFAMEtSUO) .ccscienuie aes cmeusiece s 803
Nozaki; Masumi. <:.<ssccc0. ee dd 387
Nunomura, Wataru ......................0-. 499
O
Odavint Midehors-egascacsasee eedecebe ace: 601
OSawaWNaZUONy o...cierecc6ss sscvpuetersseueisieie reverse 265, 451
OpuTo Citar ys sores cet erevavereetievsrorereicecemrevenerciores 785
ORhniShiVE WA ia ascsstvsinaee osetia 169
Ohtsuka, Susumu ......................
Ohya Moshiewcn nae. nascnscn aro eS. ctes 749
OishivKugaOiq es oscovsesteennes deeds hah 283
Okamoto, Kanako ........................5. 169
OkvmotowNaotopereeeee eee eee eee 375
OkumurasMakuyl 22 seeeesenasaea- ome ate 37
OkKuno; Makoto «.dsiciceccscccas PUES of 601
OkuyamayJunkoPeeeessessensssce see eee oe 785
OkuzawalKoichisss-c45- 2664. ate tee eee 765
Oommen, Oommen V. .................... 1055
@otsubos Wakahiro: «i205 ccccsccees Sees EE 955
O@shimaVNonkOjec cox ces cescmansockeneees eet 65
Otakin NomkOi sain deeeecateraccanar chien! 1009
P
RaoluccivyMhs 26+. seunnnicdels consents 1025
ReterpVialSalS= facet dene sskis ence eee 1055
han OeVMPAB)) ceccieteccaiasnes osiannemees 665
Pierantoni, Riccardo ........................ 365
ROMGIRMIENI SES eet iSoche oricocuscn bees eerie. 671
R
RAW BIRR atEe Sh ansanies soaeansease See 211
Rinkevich, Baruch ..................... 983, 989
IRoudebushi; WaEtierice acc sana deee eee 583
S
Saad Aibdelunt 2 .22.2ch235 2550 349, 1037, 1081
Saito, Takayoshivss...-:.:.222:h2s0ieee ates 469
Saito, Yasunori ................... 405, 983, 989
Saitou ~RaZzuo ocsccackiagans acendeoaat ena 1017
SakaguchiiBungO!s.cesmazcnee seme eeee sete 283
Sakai Masakiiegjehsrnecastchescctse see oes 955
Sakurait ShOmehiegcecenoteesc cca ieee aU 149
Sasayama, Yuichi .................2.2-0--00- 785
Satowlwanei wera ncamenncececcnect oe. ce ates 77
Sato mBakanonl: «228 nc kecana ene Dee 193
SatohiNoriyukil fee) eceee-ecesce eee cee 569
Satomi Daisulkew se Ps sccc se. ccans ones ee 127
Sawada, Kazuhiko .......................00- 803
Sawad at Momo ec siiccscicviscs tice celvene ceed 551
Saxena wy AW Kean seep eerie iis Siotreave hadron 649
SEPSIS. WAG ps eas eee a 649
Sendai Michiko sapercaceacnonee een eee 337
ShapirayiViichalueepere sa-aceee ees e sere eee 989
Sheridan MankeAwennsecens eee ese 275
1312
Shibuya, Tatsuaki ...................... 241, 515
Shimada, Yutaka) ac... Sctscrotene sateen 283
Shimamoto, Nobuo ......................4-- 749
Shinozawas Takao) ¢2:.06.sasciacce eee ones 941
Shiral- Manabu .:55.45:.0.0cstncncn elas ance eee 625
Shirai Shigeru. .....senccncsGeun st Aes ee 811
Smitty; Jimlvaylige.. bos cisco ee-noclee RCE eee 659
Sonobe; Haruyukt) ......20.2 0... aetneee eee 169
SupiE-Na@OKOM ss. kelie eee okt Sees Ae Sil5)
Suhama, Miki@n & 5.550 .cc0s2< ee BEeELE eee 89
Suzuki, AIG OW ax. setae eis s ace eR ee 149
Suzukig Kei tiie cist oc ea own cee 1009
SuzukiiMasakaziw 2.0.0: s0sacce eons Gee omens 157
Suzkie Makan Om. c.ccc ache eee eee eee 1009
Suzuki. Mats Owns scue ooeeierumee ee eee 941
Suzi, TOME 63 <.c.n0ccnectaas ac cioe ce 541
Suzuki, YuZur.,.:.nnadnedede eo eee oe 37
I
MabatayShojie cscenecceeee oes see 445
MaypmMayAideO: chs chere saps seven oteaer 1017
Takada SMichio: schccsrocsncececean cece ene 223
Makaci Wench 3 sicma veneer ace ceca ee 293
Takahashix) Kenji... oc coeds tare eee 343
Talalnagom, QUGVO oso000000000000000000- 791, 901
Takahashi, Susumu Y. ...................... 169
MakahashinMakayuki Sass esses eee ee eee eae 343
Tamanoue, Yoshiaki ......................6. 343
TanabesKobktl .)... sc.ccs see os ce dee eee 1017
Manabe, Shing: ..)..cn24sc0accc epee 193
ManakayShigeyasw 2 052k sense -cie eee a. 175
Taneda, KOjt hss cwscepsanctiee po aese eee ase 529
TanimmuraywAkihiko. see e ee eee eee eee 439
Meza, WOK Oris. icc 's.2 ose cen dee oe 619
domioka Kenji j.:iceisc oiccet asec sOeee tee 185
Tomonaga, Susumu .................... 113, 551
Msuchiya;, YUICHI) 2. oseaqcentes. ene eee 343
Tsukamoto, Katsumi ....................05- 1061
@sunekis Kazuhiko) 32.0.2... 0+... lie reece 423
TSUSUCH MOOO) .ccccosroocses ods ot nee ene 119
MsutsuinKazyOshil> cs... eee eee 649
U
Wemuraisearuko! ocecs cose ocmbitieiseeet 619
Wenos Satoshi s ees enc ontseeoee tenet cose 125
WerumovsMichacl)Vig ct aeetmet cited: 17
Wkeshima;)Attsumil ......1...).-4s5e eee 733
Wrano pAKihISal yo. csc04 000 oc see eee 157
Vv
Van Minnen, Jim. «.-......<23...0..\. sae eee 533
W
Wakabayashi, Katsumi ................. 175, 791
Wassersug, Richard J. ...................... 713
Watanabe: Hiroshi ........+.s2seee eee 405
Watanabe, Kazuo .............00.00 «pee 749
Watanabe, Mamoru ....................---s- 133
Watanabes Yuichi'\G: <5.>.¢-2 eee eee 625
Weissman, Irving, L. ....... 52 .eeeeeeee eee 989
Williams; Glynne|.-....-->- 224 eeee eee 315
Y
Yada, Takashi: 23). ...0..0....0..000e eee 143
Yamagami, Kenjiro ...............-....-..-- 891
Yamaguchi, Kazuhito ....................... 113
Yamaguchi, Tsuneo ..........: sees eee 53
Yamamoto, Masamichi ..................... 211
Yamanaka, Hidetoshi ....................-. 1009
Yamanouchi, Korehito ....................-- 223
Yamashita, Shigeki .........-..-94eee eee 887
Yano, Jumji 0.060. oe os eon ee 89
Yasumasu, Iku ....5...........- eee 329
Yasuyama, Kouji..............- eee eee 53
Yazji, Mansour E: L. ....9.ida:4seee Pee 1037
Yoshida, Masako ..........0..50. etna 211
Yoshizawa, Hideki .........:.....0¢9seeeeeeee 785
Yur Hon-Tseny osc... 0s 00s 659
Z
Zairin, Muhammad Jr. ...................6- 607
Zelezna, Blanka ............«:s;egseees seen 925
Zerani, Massimo . ....0s,....02.05 5.0 090 639
Zhang, Hongwei .................. 113, 551, 665
1313
AUTHOR INDEX
Abe, Hiroshi 1149
Abe, Shin-Ichi 1198
Abe, Takeyuki 1138
PND CAYAUIGHI © ook ds cinev nen ect te Sl MRL
Adachi, Shinji
Adachi, Takehiro
Adriaensen, Dirk
Agata, Kiyokazu
Ai, Naohiro
Ai, Takuya
Aikawa, Masuo
Akasaka, Koji
Akazome, Yasuhisa
Akimoto, Makio
Akiyama, Kazuyo
Akiyama, Tadashi
Akiyama-Nambu, Fumiko
Amakawa, Taisaku
Amako, Daisuke
Amanai, Kazuhito
Amano, Minoru
Amemiya, Shonan
Amikura, Reiko
Ando, Hironori
Ando, Hitoshi
Ando, Masaaki
Aoki, Kazuko
Aoki, Kiyoshi
Aono, Hideaki
Aonuma, Hitoshi
Arai, Junichirou
Arai, Kiyoshi
Amal AO nc.c 4 mmnyrans ninvean seas 8
Arai, Tomoko
Arai, Yasumasa
Araki, Isato
Ariizumi, Takashi
Arikawa, Kentaro..............
Arita, Tomikazu
Ariyoshi, Nobuyuki
Asada, Nobuhiko
Asami, Kouichi
1189
1200
1160, 1251
(Abstracts)
ANSATION METS AKIN 15) sara oat -aeoraichdearon HSL 1156
INSVNO 5 UO NUON) soancscssneneannoeanacde 1250
RISO AS a OME ZOE ES orercvst ss arncecrayse crsiayakenvishorevonsmottte 1154
, 1199 Asashima, Makoto .............. 1155 1156, 1199
, 1249 Ashida, Kazunori .......................05. 1192
iWon AshidaMasaakils. 5.40.5 ..0505.deeiadeeed: 1134
2S ee Atacos;moshihirojnasscse eee eee 1219
IeAh -Atysoni FelixiGinis 2c ccis sce aescere cache eee 1257
meo “Azuma, IKatsut sss ssc acces sateen ee 1230
ASG) “Azuma; Masami . 22.1... astdastebeba asks 1230
, 1242
1242
1290 :
G4 BabaeShojiWAgig.c ssa. scehonce cen tee 1218, 1219
(45; BalbalMadashigiwiee isco beso c cvine eB aeteee des 1197
leon “BawatlakakOs 7Fiscss edison vce no Se ee 1208
1138 Baba, Yoshichika .............. 1181, 1241, 1242
1295)" Bannoreloshikazu.4.-5..-254 05a 1136
4 OR Bassotewien=Mianereeeeeee eee eee 1228
WAG — iwerm, ISlOwweMGl AN, sesceecenacascnoucseccaean 1273
See Besshommomokoi-ns-c seen een eee 1155
, 1141 Bireivheide, Ruediger...................... 1251
W203; ~ Breyaule cer h cc octets s apocn Ae ANE 1134
, 1299
1188
1200 g
1283 Cervello, Milchiorre ....................... 1178
1250. “Chen SBin. & Reis cao. cc Ae ose 1243
1261 ChibaWAkirawk coccwwd oe ceecwaedetee 1257, 1274
12265 1 Ghibay Chikafumiles eee eee eee eee ene eee 1235
1249) * (Chibahomeeiek. cb occucaice os tteeee 1171, 1277
1224 Chiba, Kazuyoshi .......................... 1197
1228 Chiba, Yoshihiko .................... 1295, 1238
OT SS Chinospyukihinopeecereee reer eee ceeEe cee: 1160
pilel4G, ~—"- @hinzeis Nasu), -eAssaacseeet hs. sabe ee 1152
Id Coopers hs porcccccan tices ATBES REY 1249
mlan4 CopenhagensDavidiRe aia. --n- ce eee - 1235
1192 Crescitelli, Frederick ....................... 1230
1iSS (Currie MDouglasvAc .. cei es. coc I. EE 1271
, 1284
1301
1199 D
1133 Dan-Shokawa, Marina ..................... 1300
22S Daintdes Masashiieemeeeeeaeen eee dee 1293
1314
De Groodt-Lasseel, Marie H. A...... 1290,
1163, 1174,
Deguchi, Ryusaku
de Jesus, Evelyn Grace T...................
Diaz, Maria Rosario M. ....................
Dohra. Hideo! 2.3 .5)6ent elec’ a eee
Ebina, Yukiko
Ebitani, Noriaki
EB ouchinbisukemeereeeeee eee nee
Eguchi, Goro
Eguchi, Sachikol ...22: 2 ess.c.0 use ae ee
El Manira, Abdeljabbar ....................
Emori, Yasufumi
Endo, Hiroyoshi
Endo, Katsuhiko .....................
Endo, Makoto
Endo, Yasuhisa
Enoki, Yasunori
Eshel Dania) cvccc eke: cheer eee
Ezaki, Masanobu
Fujit; RYOZ00 2204.2 acee nck eee eee
JOH UIA, INCU coscoccccvssoavc0an00¢
Fujimaki, Hidekazu .....................05.
Fujimoto, Hirokazu ...................0005-
Fujimoto, Hirotaka
FujimotosKengoee-eee eee ee eer
Fujimoto, Masaaki ...................
Fujino, Hideki
Fujisawa, Chiemi
Fujisawa, Hiromi
Fujisawa, Hirosuke
Fujisawa, Toshitaka ....................0005
Fujisawa, Yuko
Fujisawa-Sehara, Atsuko ..................-
Fujishima, Masahiro
Fujita, Keisuke
Puyita; YOSHIAKI. 5 aciencnocn Mee aes adele
Fujiwara, Akiko
Fujiwara, Keigi
Fujiwara, Shigeki
Fujiwara, Yoshihiro ..................
Fujiyama, Shizuo
Fukada, Toshiyuki
1229,
1184,
1140, 1195,
1173,
1291
1207
1264
1155
1280
1286
1147
1251
1219
1229
1247
1133
1239
1187
1135
1164
1150
1196
1141
1302
Fukada, Yoshitaka ............. 1182, 1230, 1232
Fukamachi, Hiroshi ........................ 1285
Fukatsu, Takema’ «0.6 .c0dcs.¢0. ee 1211
Fukazawa, YUGO) «..020.5-45ace6 soe eee 1273
Fukuda, Kiyok0) 7 52..4.242 9-2 eee 1300
FukudaVut...0. 20.0040 60656 ee eee 1202
Fukuhara! Chiaki .........-...0+-- ee eeeeeee 1226
Fukui sAkimasa: ..............+0..¢s2 eee 1155, 1156
FukuisKazuji. 20... ..000 6250. See 1255
Fukumitsu, Takashi ........................ 1133
Fukumoto, Tetsuo ......................... 1249
Fukuta, Keiko) i: .....0..00. 00. --e eee 1297
Fukuzawa, Toshihiko ...................... 1207
Furudate, Hiroyuki ........................ 1235
Furukawa, Kentichi’ .......... 0-2 eee 1148
Furukohri, Takahiro ......................- 1301
Furusawa, Mitsuru .......................-. 1187
EurutaEmiko) ).5;.....+.-50: see 1248, 1290
Furuya, Hidetaka ..........-.:eeeeen eee 1281
Furuya, Sigehisa ..................... 1165, 1166
G
Gibbons, Ian Ri 24... 600..00 eee 1219
Gilbert, Mawrence I. 3.....-.5.. cease 1269
Gleadalli Tan'G: >... ...023.. 3200.06 ee 1228
Gomi! Toshiakiv, 53.4... 2.0 1290
Goto, Mutsuo 2 .......000000. oe ee 1303
Goto, Taichiro) «.sc.0.5000.0 2+ «sn eee 1294
Goto, Yoshihide: ......005...0..:0.5 eee 1267
Gotow, Tsukasa) ....<..s:000 20000 ee 1234
Grillner; Stem)... oc. 30.00. 208 oo ee 1226
Grygier, Mark Ji. ......00...00000 0000 eee 1305
H
Haga, KaZu0 onc cc eecuinje cee oe eee 1284
Haga, Nobuyuki. .....:..0.0.. 00+ see 1212
Hagiwara, Yasuko. 2... 600.0 scene ee eee 1200
Haino-Fukushima, Kazu ............. 1172, 1274
Hama, Natsuko) 2..5.)(50. .s\.0<0.00 0 eaten 1274
Hamaguchi, Miyako S. ............... 1177, 1178
Hamaguchi, Satoshi ......................5. 1196
Hamaguchi, Yukihisa .......... 1177, 1178, 1181
1219
Hanaoka, Yoichi ............... 1139, 1262, 1263
Flara AKIDIKO) \...c.cciine cise. eee 1170, 1244
Fara , ITO aI. jee s0ye.0,00ciessieiww shee ee 1294
FATA MENT Wen wink snes ene sean Bekele 1195
Fara. Masayuki ..:....0scc-eeeeees ee eueeee 1138
Marada\, Hosamil :.<..2..:..6ie.. Sa 1285
lama aU SSClt ion el siete snvec asc ics ome ete oe 1146
laradatMetsuO: «oc. caccswsce ands eeetucke 1252
Haraguchi, Hisashi ......................... 1260
Iancayay Voshio® .....¢022)26 1.2 eh tas. daee 1274
Harigaya, Wakako ...................0.008- 1138
Hariyama, Takahiko ................. 1233, 1238
Haruguchi, Yoshiko ......................5. 1165
laruimibeatSuO) . 22.22. .sccascescen ne? 1204, 1205
Iasegawas Kazu. ..ccevec ec eeeeece ee es 1195
Hasegawa, Sanae ................... eee eee 1257
Hasegawa, Yuriko ................... 1135, 1272
Hasegawa, Zutaro .....................008. 1155
Hashimoto, Koichiro .....................5. 1172
Hashimoto, Naohiro ....................... 1169
lashimotos YOKO :<..issc0< codec. sees ce lade 1236
latanakaewlsune@) s..0...s50+s+o seen 1245
Hatsumi, Machiko ......................... 1188
Rlattamshinicht. 2c. .sccccccccccds sss: Maes 1228
RMATCONIPINCISUKE? ocgc5 ccc oo hae cote cosa ee 1216
Hayakawa, Tsuyoshi ....................... 1175
Hayakawa, YOICHI. ....:0..s0s.00s. 5. sites. 1270
Hayakawa, Yoshinori ...................... 1294
Hayashi, Hiroaki......... 1139, 1261, 1262, 1263
Hayashi, Hiroshi .......................005. 1165
Ifayashindinoshi) 22. 4.6.262-c.2-22s sees ees e- 1229
Hayashi, Kensuke .....................-00- 1200
Hayashi; Mikal io. .20se%0ct ere JES 1239
Flayashi,;Shinjil ©... .sceeeeees cect ees wate ee es 1265
Hayashi, Susumu ..................2-e-0 ees 1294
emine Peter dn te). c2c2ccorn-kee teense ote 1230
irdakay Soh (Aes oh eek ssn dc AGRE oR 1236
irdaka OOD: s.5 sa0 088s has cee PRRs 1239
CAA VONKIW ES a2 s22500c0c07< cde RGR 1202
SUCH EIdEO . ascecaceres vere nc Seale . 1147
Higuchi, Takashiro .......................05- 1241
|ShiqoriNe), Iti $0) oooadeneoauevseopodobogceauE 1200
Elikosakas Akira! 2h) Qocccc cc ciose ccc se das ess 1193
PLTVRUISSE IME Ms aed otis von ee ee 1226
HimmuanasMasaon ss. s2.c5 0c. . soho tenn scene 1277
Hino, Akiva)... 22 hence cee cacw nee llay! 1181, 1303
TINO GAY OMe nadine. d ode c vig Maas oe 1147
Hirabayashi, Tamio ............ 1141, 1160, 1168
1169, 1173
lebimil, IENGMERS, Gosoooscdnodouspanooneeseoe 1181
irai® Moshiaka) s-secccec sek p cole cede fhe 1162
Hirakawa, Noriko ....................0 eee ee 1280
Hiramotoy WUKIO Geek eos ancsesces Se ha 1177
inanoW Hiroshi. ss<sccnccasecnss bien sees ee 1250
Hirano; Husadshit2. ..4cc0h02 eee: 1151
PInAanoWNenzichil .cce 2 ene ceed ase ees 1175
LBUTEIO), INGIINK) Gocnaaoseg0sd0d0 009006000006 1210
JBI), WSISUWVA, sancooscsnasagvang0ns 1257, 1264
PLiratay A UNKOM AME coscncotecnee es Sees 1203
FinobesMomohisaencess2. 4.2) ee seen nae 1208
Hirohama, Tohru .................... 1260, 1261
Hiro Tomoko ewes saan cease eee 1138
Hirono, Masafumi ................... 1148, 1212
PirOSe ME Wich. 6.-ecaeeee eet 1207, 1213
lino taeyKGyOnonileasee--eacecee ee eeeeee 1151
Hinukawas Yukiko). .ces..0s0: 5: see eee 1277
Hisada, Mitsuiko ..................... 1224, 1294
Hisanaga, Shin-ichi .................. 1161, 1176
Hdisatomit Osamu 2------..+s0s- one ee 1231
Hiwatashi Koichi.......................200. 1279
InlolllniOlel, VOO Ga cassccosessvscscsccusucoac 1233
JEONG, IGUIROT sas5onascsncasscobedaodeoooc 1253
Honma, Yoshiharu ................... 1257, 1274
Fel OTIC SKIP cae cs cake ey 1215
FA OUIUVISA Oia sete O58 ice notre teas NR 1185
MonyManabuleh: ccccccacccsscccs Ane ee 1212
ionkaminbhidekitesrte seater ee eeeeeee nee 1277
FHOnuchIsShinnieessseae eee 1182
HoniuchisShinoe h.sc0ccesc<cc sss Re 1139
Hoshi, Motonori ............... 1175, 1197, 1304
HoshidesNazumilsss--2ss esse eee 1297
Hoshino, Katsuaki ................... 1204, 1205
Hosokawa, Kazuko ........................ 1179
Hosomi, Osamu 05 .0d0020<05 2205. bee te 1139
Hosono, Masamichi ........................ 1247
Hosoya, Hiroshi ............... 1143, 1145, 1146
Hotani, Hirokazu .......................... 1161
FOU ROUIOE seen oases 2 ace Se 1137
Hyodo, Susumu ..................0..00 eee 1267
Hyodo-Taguchi, Yasuko ................... 1287
I
Ichikawa, Masumi ......................... 1289
Ichikawa, Toshio ....................0..000. 1236
IchinosemElinoshiesssss-454.05 seen ee 1135
Kdetnhiroyukiv ere. aeeaec este tyerds OIA 1201
Kdeyama‘ Shine 3: x55 s3asnesce0dase. Oe tee® 1247
(Ge\, INGHSUNO) ooeaeoconsenoegenaue 1226, 1227, 1228
1316
Igarashi, Yoshihiko ........................ 1206
Iguchi, Taisen ............ 1265, 1266, 1267, 1268
1273
lhtarasSetsunOSsukeerer eee EEe Eee EEneree 1202
[hara’, SHIN aes sacucn saeco ce cee eae 1260
Tijimias AKIKO. coc sngeecieoe te ee ee 1292
Tinos, TeRWhi KO... osicaecies cee eae < SA ee 1135
Tjime ShigehO)aceecenicce sone onemade eee 1162
IKE DE, YUKO a eccenche esis osc oe Re 1296
Wed axtAkinaey . ccscc:5coecn once eee ee 1225
Wkeda. Manik) csc. osccuacpcucnson eee 1158
Ikeda wMiasaukil, ...c.-c06. cer eee eee 1238
TEM a IVITE OF oysiorvans cise bees re SOR 1270
ikedarslietsuyalasa-reee eee eee eee 1223, 1251
Ikegams Masato) > 3-42.20. ..ceeien eee 1143
Ikegami, Susumu .................... 1167, 1179
ikenishityWohijiteeese ee eee eee eee 1173, 1187
Ikezaway Hiromi 425-2 ee ee eee eee 1304
Ikwita, Kyosuke® ..c0% coe nassau Seen eee 1283
ImatukuyMichioy--- ese e eee 1305
ameArSeaNyiel, IMUINO) apecccaccaaaccgca000ncnocoac 1219
Imai. FinOOmiciees doc ute canon oA One 1230
Imai: KiyohitOs.j.cced.essnaseceste ee eeeee 1252
maby as KWaZu@) m.ccacicenis nc ceneee ee 1145, 1206
Inagaki, Masaki ................ 1143, 1145, 1146
Inamori, Megumi ....................+---5- 1300
Imamura, Hiroko ........................00- 1282
Inamuras©OSamlieeeee eee eee eee eee 1228
Inase SWOSMINON. ae. a5 ctisoe oo no RRO 1254
Inohayar Neils fete een eee 1157
Inokuchi, Tomofumi ....................... 1139
Wao, SRYGENKO) 55 000c0nscagns0n¢onacnn 1173, 1174
INOUC>, YASUO! 2665000 se ete eee 1173, 1174
Inouye Shinitidy .3.2 50.02. sheer ee 1226
TS@ka A CHINOW Rye a.c, os ts ssecnsicnee SOREN 1203
Ishibashi; Nakaakiy).... 0+ +.c0- eben 1242
Ishibashi, Yasuhisa......................05- 1277
TshidanAidekiies «..c:0:0¢-s:csevorw oi re 1227
Ishiday Katsumi) <i. see ae ee aoe 1220
Tshid arias ails ovrevers vs ecoie erare sr oeseern ree 1143
Ishida, Sachiko ................. 1184, 1185, 1186
Ishigakik Reni. ccc. s+chiseseeeeoeeien cen 1252
IGavlnene, Inbbiol0) Gaananconaqungcocsnncncncos 1289
Ishihara, Katsutoshi............ 1160, 1164, 1165
Ishihara, Mitsuaki......................000. 1148
Ishii Kei-i1chiie facts osc rs ai noses ar 1277
Tshiith, Nort Oi siete cic nice cic cies clacetare ov ee 1144
[shite S ADU nO Meera cera esstaed makin claw cranes 1282
Ishit.eSuSUMMUlGe eee oe oe eee eee 1266, 1293
Ishii, Téruhisal cats... s2eces ss ee 1176
Ishijima, SuMIO\;..... 22+. ....-5eeeeee 1219
Ishikawa, Hajime ........ 1152, 1153, 1211, 1285
Ishikawa, Kyouko ..........:..+.-2=ee=e—eae 1281
Ishikawa, Yuji. .....+2..:..::+ Seen eee 1287
Ishimoda-Takagi, Tadashi .................. 1147
Ishizaki; Hironori .............2oee eee 1269
Ishizawa, Nobuaki ......................... 1181
Ishizuya-Oka, Atsuko ....................-. 1204
Tsono,: Kuni) sicceise. 2s: on oo ee 1231, 1233
Tito, Kazuo: ies. oo. cece senses. oe 1208
Tto; Masamichi »)3....5........... eee 1297
Tto,{Masayoshi ..2.:...:....+2..-2eee eee eee 1231
Ito; Yasuko) 22: sehe. + 20.200. 0 ee 1270
Ito, Yoshiyasu) .ic0..... 2. «ee eeeEe enone 1200
Tto,) Yutaka 2 ccss0: :2..55 26. eee 1150
Itoh; Keiji: sas... os... e200 co eee ee 1187
Itoh,,Masaharu’ ...............0eR eee 1147
Itoh; Tomohiko) ..............+. see eee 1161
Itoh, Tor. . .c..c060.005 osinns eee eee 1199
Itoh, Zen. ....is08.0500000000 eee 1284
Itow, Tomio............0..>.eeeeee eee 1154
Tuchi; Ichiro, 8 ....50...420...ese eee 1157, 1171
Iuchi, Yoshihito ...................... 1163, 1164
Iwahori, Nobuharu .......................-- 1289
Iwai; Yukiko:.2 .. e.0 s0c.++.0 + oe ee 1171
Iwama, Akifumi ..................... 1240, 1244
Iwamatsu, Takashi ....... 1160, 1170, 1173, 1209
Iwamuro, Shawichi......................-.- 1262
Iwao; Megumi... 2.2.62. 2.0.+ oe eee 1156
Iwao; Yasuhiro ........... 4700 eeee 1161, 1206
Iwasaki, Masayuki ..................-...-5- 1233
Iwasaki, Naohiko ...........:..+sce seen 1230
Iwase, Tomoko. ........ 6.0.0.0. 000 dee 1159
Iwashita;, Shintaro).....0..... se sseee eee 1169
Izumi, Susumu.............++++++0. eee 1141
J
Ji Zai-Stwsiwee occ sncrcanaasis eee 1198
Jikumaru, Shouta ...................0000eee 1206
JinguhyipYouichiss..... 2.55... 0-5 1150
Jongwatiwes, Somchai ...................5 1211
Juchault, Pienre! .... o..00... 05. Ge ee 1135
K
Kabasawa, Hiroshi .................000.00s 1225
Ieado tas MEtSUO) .iojectcce science ta ol 1225
Reapawval IUTOMIKO oi... ee ee ote 1257
Kageyama, Tetsuo ...................e eee 1175
Gain aAZUMIKO) oe os cect ote weve dee de ae 1278
Reap imassITOKO: 0.60. jee ee hee ovate 1162
Kakeyama, Masaki......................... 1255
IRENE NIG AS) 00) eS Oe 1257
Rev PAU eS is corre eee 1250
Kamata, Yasuyuki ............. 1165, 1166, 1167
CAMS WARN ORWMA «pce ese ace HA Rae AE 1273
Kameyama, Masaki ..................-.---- 1234
Kamidochi, Mika .....................0005. 1215
Kamimura, Ryuichi ........................ 1210
Kamimura, Shinji ..................ee eee eee 1144
Kamishima, Yoshihisa ..................... 1227
AMM aA SHINO Na sisi ca ee eee See cl 1279
iene. (Qe) <0) tag aoe eee Banh 1190
Ream atSUakans .... oe: se wad ce ee ol IER 1288
Kanbarasirojil sco... de taen ddl, eeu. 1211
Kanda, Masahiko .....................00005 1160
Kean al Miyuki. 2. cece cae eee ADA MEMABles 1166
KReamd a mbOshiOne:. ..0cccee een wn otidhicles «Mae 1270
Ream KOM MASA OY ce ecie a cieressnscvoirrrcseue fein ees Shae 1176
Kaneko, Nobuaki ........................0. 1206
Kaneko, Tomoko .................... 1208, 1214
Kaneko SOV Opii isch... cie cece ea ee cee bale wd 1257
IAMS KOMIK ONG ccicjeeicice ae ereve es ehhleldelele ott 1182
Kang, Wonkyung ....................00005- 1141
Kani-e, Toshihiro .......................00. 1160
RAMON VCASWIATKO! 5d. oye ccis iors oie ole bleclatetolnn «Sle Bie 1202
Kanzaki, Ryohei ..................... 1225, 1240
Kanzawa, Nobuyuki ....................... 1147
Karakisawa, Hideyuki ..................... 1232
Reary aR MISK OY Sei e ciesee ele en Mali Maile 5 wattle 1137
Katagiri, Chiaki ...................... 1180, 1199
IKgrizweatal, (Clive) Gagoseedsso0000G0050405000c 1142
Katakura, Yasutoshi ................. 1135, 1272
Katanosaka, Kimiaki....................... 1231
Katayama, Heizaburo ...................... 1228
Katayama, Noboru ......................05. 1247
Katayama, Tomoe ....................-005: 1302
Kato MKOehin sensei. sno bie EL 1179, 1181
Keaton MakashiQ' 252 0.0cccccicsles oe BORER 1267
ATOR KY OPPS eet osie neresiesaae ae daa Gch 1263
Kat OMIA SKE give recs. roreibne dhe teehee 1172
Kat OW MIKA OMG aso: esis ereceie ordiaie w leas wo ohd Boek 1221
IKETION, IMBVHIKO) -5555050000000000000005 1212, 1151
KatOMsSEtSWKO GS icc: chscivsnciecespuocso ey S Ole dee 1135
Keats usPYOSNIMN AO sits. acs: ccenovencsoccd hap eh SANS 1162
Keats um ViOichi sei ecccces ncameataeeoden 1134
Katsura, Yoshimoto .....................00. 1247
Katsuyamial, YOuI miner aces. acsscdeeriered- 1195
AWA SOKO NSS S oars. ela iiaceneics TR a Ae 1208
KWawalharas vA Kia). soca erses caddis dete Melde 1203
Kawahara, Masahiko ...................... 1228
Kawahara, Shigenori ....................... 1240
Keawiai Miia ISee ooo salocmats ona one do 1299
Kawakatsu, Masaharu ..................... 1298
Kawamata, Kunihiko ...................... 1234
Kawamoto, Manabu ....................... 1166
Kawamura, Kazuo ................... 1195, 1196
Kawamura, Ken-ya ............-.......0-5- 1181
Kawamura, Kosuke ........................ 1263
Kawamura, Yuuki ................... 1146, 1147
Kawasaki, Ryoji .............0.: eee eee eees 1238
IRGWWERAl GL, Were ooccnccncncnnaceoonsucsee 1261
Kawasaki, Yukishige ....................... 1184
Kawase, Eihachiro ......................--. 1172
Kawashima, Selichiro ................ 1254, 1258
Kawashita, Hiroto ......................... 1143
Kawauchi, Hiroshi ......................... 1257
IXBNEL, IMERETS Ch cis Gee aeee Geers oxtbise Bet olor 1268
KeinosHiroomiliaseew... . os. 00 ee A 1136
Kettoku™ Masakomeee =] eee eee Eee eneee 1166
Kikuchi, Shin-ichi .................... 1249, 1288
Kikuchi, Yasuhiro ................... 1290, 1291
Kolkutasoshitenigeeeeeeeehceene eee eeeeee 1289
Kikuyama, Munehiro ...................... 1177
Kikuyama, Sakae .............. 1139, 1255, 1261
1262, 1263
Kimura, Akihiko ..................... 1290, 1291
Kimura, Kazuyoshi ........................ 1211
Kimura, Ken-ichi .............. 1189, 1234, 1271
KoimuraZKOUKL siicccos cee cine ke ee oe 1280
Kimura, Masashi ........................... 1212
Kimura, Sumiko ..................... 1146, 1147
Kamunal Pakeyit ce ec ncrnaceome eee oe. 1293
Kimura, Tetsuya ..................... 1241, 1244
KinohMHinoakat so. ode scen sles 1205
KimoshitawEijit ccc ce aceace secs occa te alseh 1288
Konoshitatikeiyya.. .as.4.-.5.-42 eee 1156, 1159
Kinoshita, Masato ......................000- 1209
Kinoshita, Tsutomu .................. 1156, 1159
Kirinoki, Masashi .......................... 1168
KaiShie KiyOshie. i cesieasces temeees ace 1290, 1291
1318
KishidasAkihiroiees ee eeeeee eee eee eee 1255
Kishimoto, Koichi ......................2.. 1161
Kishimoto, Takeo .............. 1142, 1161, 1176
Kishimoto, Yasuo ...................00000e 1183
KoitaryNobuyukiteneeesere nee eee eee eee 1179
Kitajima Ken 2c cnc cmcnnc ones ceeeneee 1173, 1174
Kitamunatsumieceeeeeeee eee eee 1189
Kitayama, Tetsuya .......................5. 1246
ISO, ON eo eee enh anadoobodode ar 1298
ICOM GU esoa ee cack nvan eee ee 1228, 1230, 1252
Kiyomoto, Masato ................... 1158, 1159
KobanmhumikoOpere sss eee eee eee ete eee 1247
Kobayakawa, Yoshitaka ................... 1190
Kobayashi, Hiroaki ........................ 1182
INobayashis) Kant=-----eee ae eee eee 1203
Kobayashi, Ken-ichiro ..................... 1139
Kobayashi, Makoto .................. 1222, 1223
Kobayashi, Michisuke ...................... 1252
Kobayashi, Michiyori ...................... 1252
Kobayashi, Minoru ........................ 1152
Kobayashi, Satoru ................-...-.-5- 1188
Kobayashi, Seitaro ................... 1185, 1186
Kobayashi, Takaaki .................. 1137, 1145
Kobayashi, Takanori ................. 1300, 1303
Kobayashi, Takuro .....................0-0- 1197
Kobayashi, Tohru .....................0000- 1256
Kobayashi, Wataru ....................-00- 1181
Kobayashi, Yasuo.............. 1259, 1260, 1285
IX@ RWI, YOUU 54550009000 s900ac0c0n0g0000 1216
KodamayRyujlj eee eeeeeeeeee ee ener eee 1189
Koganezawa, Masayuki .................... 1246
Koo, SHirOshil.).. ...nacnenin eens Coos 1254
Kohama, Kazuhiro ......................--. 1147
Kohda, Yasutoshi ....................0..05- 1288
Kohno, Hiroyuki ......................0 eee 1138
KOtke Satoshi. iis wei wowona wae Mee 1268
Iowan, SOS WO s55009000s0950000000005000- 1134
Koizumi OSamulyee sete eee 1190
IXOpunne. IDEN 3555500000000 90000050000006 1230
IX@WRTEL, IMMNENS! G55005500200000G04Gu050000e 1269
Kojima, Manabu K......................065 1181
Kokubu, Tomokuni ........................ 1143
KeOmatsu VARIA 5.5.65 oscr cause nce wie arbiters 1233
Komatsu, Miéko ..................... 1292, 1299
Komatsu, Satoshi ....................00eeee 1143
Kominami, Tetsuya .....................00. 1178
Kondo TOshih Se sececmie-e oeeeeeete aaee 1286
Keon OM IUIN oasis vis dro.sisie Veewreciowae an Mes 1253
Kondo, Noriaki. ............5... 0222 eee 1253
Kondo, Yasuhiko: ..............-- ..eeeeeeeee 1255
Kondoh, Yasuhiro .......-.... 2p eeeeeeeEe 1243
Konishi; Kooichi:......... 012) 2252 eee 1305
Kosaka Toshikazu ............. 1276, 1279, 1280
Koshidas, Yutaka .........--..500eeeee 1281, 1182
Kotani, Susumu). .....4.5)..... eee 1150
Kouki, Tom: sic 52.00.02 000000 00 e 1261
Kouyama, Hiroshi ......................... 1138
Koyama} Eiki Wis:40c......:.5 eee 1201
Koyama, Hiromichi ........................ 1191
Kubo, Toshiyuki. ..........:.-:+e Reese 1210
Kubota; Hiroshi ............)..sseeeeeeeeae 1175
Kubota; Ichiro. <.. <2 ...¢5....0. eee 1223, 1251
Kubota; Shin ..........-.0.... «eee 1190
Kubota, Yasuo .................. S¢:esepee ee 1208
Kubota, Yukahiko .....-.-- sense 1208
Kudo;, Hideaki... =. ...........0..:00 1008 e ee eee 1244
Kudo, Shigeharu ........................... 1170
Kumakura, Masahiko ...................... 1203
Kunioka; Hiroshi’ .............- eee 1239
Kuno; Shinnichi .........5..-s. sees 1168
Kurabuchi, Shingo ................... 1203, 1261
Kuraishi, Ritsu .........:...0..% cee 1158
Kuramoto, Taketeru ................. 1216, 1217
Kurasawa, Yasuhiro .....................-. 1151
Kurita, Takeshi, .......0%.00.0.9t2. see 1286
Kuroda, Hideyo ..................... 1175, 1206
Kuroda, Masaaki .................... 1149, 1150
Kuroiwa, Atsushi .................... 1200, 1201
Kurokawa, Makoto ........................ 1223
Kurokura, Satoshi ............. seen 1210
Kurosawa, Yoshikazu ....................-- 1213
Kurosumi, Kazumasa ...................... 1261
Kusakabe, Takehiro ................. 1140, 1193
Kusakabe; Tatsumi ...:......0. scenes 1290
Kusano, Kazuhiko .............05en 1239
Kusano, Toshihisa ......................00. 1211
Kusada, Hidenori .......................... 1206
Kusumoto, Naoko ................-.20e005- 1270
Kusunoki, Sinichirou ....................... 1228
Kuwagaki, Demmi .......................4. 1262
Kuwahara, Tsutomu ....................055 1185
Kuwasawa, Kiyoaki ............ 1217, 1221, 1222
1223, 1241
Kwon, Hyuk-Chu .......5.....s0. note 1259
KwanyO-Yuw ec ccan cesses yctee ce 1152
Kyozuka Kel-ichiro ..................0 eee ee 1174
L
WECM ONG=SU yoo. ce eee ese e eee eenmeuebens 1157
M
IMabUGMIISSE1: bs. s1e5c6 cc lesa eee 1145, 1207
Machida, Kazuhiko ........................ 1137
Ifa Ini aya 0) oicscccssacscasensuorovevstererave. spol diate ove 1254
Maehara, Michiyo ....................0000- 1236
Maekawa, Kotano: .....0..6ccewe cd ee bee sce 1198
Makabe, Kazuhiro W. ......... 1140, 1192, 1193
IIT ANKIKO) ess svisenscoreniareens nt Sede ct 1190
Makino, Naoya ..............-. 1282, 1284, 1196
Makino; OsShimanl ....00..2000.00s000. onsen 1240
Makioka, TOSHIKL 0.05: cece ee ble pelele ore 1283
Mamiya, Yoshitaka ..................0.005. 1270
IMfamabe BAC aS. byercie:.-sverecoreirere Sila ewe aay lee 1147
Mandelkow, Eckhard ...................... 1144
Marikawa, Yusuke .....................0005 1192
NMilatiny Gilli yee os. ececeysyeatersrersre ADEE AS 1135
Maruo, Fumiaki ..................... 1171, 1182
Mar O Ka MINN ON Ais. cseseccsrece scare esas clvioare ales oxsbe 1159
Maruyama KCI 2 )s6.c.)--eicresee-coe Hedbek s weellal 1138
Maruyama, Koscak ............ 1146, 1147, 1148
Maruyama, Tatsuya ........................ 1287
MiasudarvAtsUkOs cs.) csochearccne Shee. Manas 1267
IMe@RUGE), Jeli <C) Seeeeonencoonmacasaoeeeone 1233
Masuda, Ken-ichiro .....................05. 1133
Masuda WOSHIKA: J.)..c10)vorcren reer homie tlhe 1305
Masuib Akaynakal ais orceytercrcrerssem betes bic chttaees 1176
Masumoto, Takumi ........................ 1270
IMGUNAL, ISUTROS MT saccsengosneaanvcsc0nuc0enc 1213
Masuyama, Etsuko................... 1143, 1144
Matranga, Valeria ......................-5. 1178
Matsuda, Kouhei .....................00055 1262
Matsuda, Manabu .......................05. 1258
Matsuda, Motoko .....................00005 1188
Matsuda, Ryoichi ....................00000- 1169
MiatsudabeSeijiie et biti: jee nrc SOE ee 1279
IMEKSICA, NAIVAIIAN (Sp agbeonde doubdodooseosaes 1146
Matsuguchi, Shinobu ....................+.. 1186
Matsakis YUU tities ccasessnieasers Seewelse ous 1239
Matsumoto, Akira ............. 1194, 1267, 1295
Matsumoto, Fumio......................... 1293
Matsumoto, Gen ......................0000e 1151
Matsumoto, Isao) s....2...4222+ dee een. cee 1180
1319
Matsumoto, Jiro ..................... 1207, 1213
Matsumoto, Kiyoshi ....................00- 1276
Matsumoto, Masami ................. 1197, 1210
Matsumoto, Midori ........................ 1304
Matsumoto, Yoshihiro ..................... 1167
Matsumura, Akiyoshi ...................... 1288
Matsumura, Shinji ........... eno So 1221
Matsuno, Akira ................. sooos WIS), 1226)
Matsuo kal Arka i). oycsccetcvesovorerere oldetiodee's adaiee 1136
Matsuoka, Norimasa ................. 1299, 1300
Matsuoka, Tatsuomi ....................... 1224
Miatsusakal Tadao: ric c.ssvvre se bbmners v oheeele 1277
Matsushima, Nami .......................-- 1270
Matsushima, Osamu ................. 1223, 1251
Matsushima, Toshiya ...................... 1226
Matsushita, Atsuko ........................ 1232
Matsushita, Susumu ........................ 1195
Matsuura, Katsuyuki ....................... 1175
Matsuzaki, Morio ......................000- 1284
Mawatari, Shunsuke F. .................... 1304
Mazak Say uichity sireiysc ates vec dee oete dt 1198
Meyer-Rochows Ve a..ch aces cine nnd seeor< 1238
Michibata, Hitoshi ................... 1250, 1251
MichidasEinroshit@ene.s.. 4542s anes 1261
Michikawa, Yuichi ......................... 1197
Michinomae, Masanao ......... 1230, 1233, 1252
Michio, Souichirou ......................... 1137
MifinemKkumatoyeeeee eee enter eee 1232
Mikami, Kazuyuki ......................... 1275
Mikami-Takei, Kaori ...................... 1286
INITKIBRW ata RUDE 3. 5/sssseccteweis.archoxes SERRE 1223
Via VAI OY eset cvaldacseysts-uersraresyiosi UO Eo 1192
Miki-Noumura, Taiko .......... 1143, 1144, 1159
Minakata, Hiroyuki ........................ 1223
Minami, Narihiro ........................-- 1149
IMPINGE, SUITWKO socecccccasacnsecnccodbucnc 1190
Minokawa, Takuya ........................ 1299
Minori, Nobuyuki .......................... 1248
Nf See yIN ia thames erearneyscscasysneesae SPARES Ao cee 1154
IMO OVATEL, SUOMI ococcccccccoccncscousuoouer 1278
Mita PIRO Ui chi ysevicerctsesnexcisceranivs RAAS Soh Oe 1199
MitaayMasatoshitaeerrncceceicn canner ee 1161
Matar aA SUK oho oss eveiseciesevevsns: bt oa 1172
Mitsui akas hile 4. sxjsvsrccveron dbs dines helen 1189
Mitsunaga, Fusako ..................0.0005. 1265
Mitsunaga-Nakatsubo, Keiko ........ 1143, 1166
IVA TeeAP ECS I gts eee is con vcncncvaysnsns ORT ence 1152
Miurasaakeshi@enanceeeeceeerernoe eee 1256, 1199
1320
Miwa, Isoji
Miya, Takahito
IMO, VEISUINIKO) pscocescaod00c0ccuag00000%
Miyagawa, Kouichi
Miyakawa, Momoko
Miyako, Yumiko ..................-2.2.005-
Miyamoto, Sio
Miyashita, Yoko
Miyazaki, Jun-Ichi
Miyazaki, Katsumi
Miyazaki, Tatsuya
Mizoguchi, Akira
Mizoguchi, Hazime
Mizoguchi, Masako
Mizukami, Atsuo
Mizunami, Makoto.........................
IMBVAUIBYO), SYMON MI 5 ococcco0ccngccnccousencnens
Mochida, Kazuhiko
IMC MATS, IRGIKO occnccccncvc0cnnc0csescene
Mogami, Yoshihiro
Mohri, Patsuitialessscicscrcerstveeusezasco tee eee
Mori, Takao
Mori, Takayuki
Mori, Yasuko
Mori, Yutaka
Morimatsu, Akeshi
IMIGTAOVKAL, IMEVAU® oaccccscnnnecuncocsscocudees
Morisawa, Masaaki
Morishita, Fumihiro........................
Morita AKInIn©) 2:2,ccsccccnc. Pht eee
Morita, Toshiteru
Morita, Yukitomo
Moritaki, Takeya
Moriwaki, Kazuo
WOTAHVAL, INUINEO) cococcocecococcnccsconedonce
Moriya, Megumi
Motobayashi, Yumiko
Motokawa, Tatsuo
IM Bias YASUO ii sescctossscnsieseumeserscensan eee
Mukai, Masanonil < 252.0000.
Muneoka, Yojiro
Miurtachis Shino taies <.c..issicerpavesins Oe
Murakami, Ryutaro ............
Murakami, Shizuko
Murakami, Hoshi ka): o.:5.0 ciscsese eecielen a nantes
Muramoto, Atsuko
Muraoka, Hiromichi
Miuratar seni cic meusctianmeecacoeieeer ac
1142,
1173,
1213,
1228
1141
1283
1200
1153
1229
1217
1232
1259
1151
1250
1254
Murofushi, Hiromu ................0: 1161, 1207
Muto Yuitaktaiieciscc: osc os oe <o eee 1150
My otoishii YukiG=---- eco eee eee 1179
N
Nabeshima, Yo-ichi ........................ 1169
Nagahama, Yoshitaka.......... 1256, 1161, 1162
1199, 1274, 1278
Nagai, Kazuo. o.oo. neon 1252
Nagail Wakatoshi 2: ..--.--.-- eee eee 1244
Nagaishi,; Hiroshi ..........:..-..2eeeeeeeeaee 1227
Nagamoto; Jun! ... 2.0.67. eee eee 1294
Nagasawa, Hiromichi ...................... 1272
Nagasawa, Hiroshi .......................0- 1267
INagashimas Kel |) 02.7.4 cee eee 1250
Nagata, Kobi ic ecicdee aoc eee 1269
Nagata Saburo) <c..----+ eee ee eeneeeeaee 1259
Nagatani; Harumi ...........7-) eee 1231
Nagatsu, Toshiharu ........................ 1135
Nagayama, Toshiki ........................ 1224
Naguray diakanor 22... e eee eee ee eee 1168
Naitoh! Tomi: .2:....:....05....0.+ «00 eee eee 1246
INaitohs) Yutakay force eer eee ee eee 1218, 1221
Nakagawa, Azusa ...............c0ceeeeeuee 1240
Nakagawa, Hiroyuki ................. 1148, 1149
Nakagawa, Takahiro ....................... 1167
Nakagoshi, Motoko ........................ 1135
Nakajima, Haruko .......................-. 1181
Nakajima, Keisuke ............. 1261, 1262, 1263
Nakajima, Yoko ..................... 1158, 1179
Nakajima, Yumiko .......................5. 1216
Nakamura, Akio ...................5. 1140, 1174
Nakamura, AKiO .....)....... ..0%..00 eee eee 1301
INakamumasAlkiray 5: c...0-0 eee 1208, 1214
Nakamura, Biko) ..,..........-.--: eee eee 1146
Nakamura EdeKi 0.2 seen 1224
Nakamura, Hiroaki .................. 1249, 1288
Nakamuray JUmt: 2 ....<..:0.0:8.0,0.01,0:. eee 1142
Nakamura, Ken-ichi ................. 1143, 1144
Nakamura, Masahisa ....................... 1197
Nakamura, Motonori ................ 1216, 1217
Nakamura, Satoshi ................... 1185, 1230
Nakamura, Shogo)............... eee 1181
Nakamura, Sumio ..................-. 1260, 1261
Nakamura, Tadashi .....................05. 1245
Nakamura, Takanori ...............:....00- 1156
Nakanishi, Akino ................0.ee eee 1239
Nakanishi, Yasuo ................0e eee eee 1202
INIB SE 1X0}5N B1Y40) Bes a ee eS 1178
Nakano, Hiroshi ...................0cceeeee 1155
Nakano Shiho) ..ascae.cesseeee ieee Sits ot 1147
Nakano, Yoshikatsu ..................00005 1292
Nakao Kyoko: 2.2.0.0... ccs eee eee eee ees 1232
Nakashima, Seiko ...................000 000s 1170
Nakasone, Kunihiro.....................05- 1287
Nakasone, Masato .................00000005 1167
INakattamit Kei .6iisccneine weenie eo ees 1234
Nakatomi, Kazufumi ....................... 1259
Nakatsuji, Norio ............. 0c eee eee eee 1172
Nakauchi Mitsuaki ................... 1301, 1303
Naika chs YAM. c.nccs cence. hee etele 1146
Nakazawa, Shusuke ......................-. 1211
Nakazawa, Tohru .................... 1138, 1159
INamibaeSsSaakt ..ccccccacron cae suneste coe ee. 1224
INgVinlOUL, ZAT0) Aa eee ee ema 1140
Namikawa, Hiroshi .................. 1190, 1304
Namiki, Hideo ........... 1137, 1227, 1286, 1289
Nanita, Kamya) i... 20. cence ieee cee es 1230, 1252
INARUSEHISIVOKO: csrctcaneiaiuicnrends ore eees 1261
INamuse) Mayumi Genie eccl nce se eee eee 1296
Naruse, Mitsuhide ......................... 1261
Natsume, Tsugitaka ....................-05. 1166
Nawata. Homoki ....05....0... 002000 sees 1187
ING AROMOK OMIM sivjareiistentecrettoneten Soh 1141, 1270
ING oiShis SumMIkO cyte ci ariel dle elne ee 1135
INGMOLOMINORIKO® joc aceccoccceeere slo ee) ce soe 1273
Nemoto, Shin-ichi............. 1177, 11778, 1181
Newland SPhilipyl, jc cece ccs secre ne: 1243
INidaWAKIYOSHI icc cetreuieie se ed eels 1243
INGO, MEV IKE «cer. rrrcreinetereigceteerre doe eea ie eae 1235
INIT NGUUZ0).3 AA te ne eine kt ate ae OLE ar 1171
INS Oi, ERUPC0) Cece eee eee 1293
INS Fata AKA OW 5 i fz Staves ge cereionsycicieeelenoversusrsvervorane 1234
Nishibayashi, Sakiko ....................... 1260
INisShirda, SE ISUKE: <ssys¢..r.5te;ornenervcmre. Seaetownens 1150
INishidaliSachivOirerr.- peer erento ae 1180
Nishido, Wakana ...................0..000. 1254
Nishihira, Moritaka ........................ 1293
Nishikata, Takahito ........................ 1192
Nishikawa, Michiko ........................ 1242
Nishikawa, Sumio ......................000. 1203
INishikawas Menuaki) Wr)... aeeeieeee - 1303
Nishimaru, Matsumi ....................... 1216
Nishimatsu, Shin-ichiro .................... 1156
Nishimura, Kenji .................... 1196, 1214
Nishimura Mani aaeceeeeeeccoe on een ee 1149
INIShinOMEInOSHIO 44. aaa csneonne seen. 1225
INShrO Kae IMG OTI. a erervowerccsac rece Aeeee aon aat 1302
INISHIWAKIS YAK O) co sacicrsavexresevsie o ootlcoen neces 1182
Nishiyama, Ichiro ..................0.00000: 1183
Nishiyama, Norie ..................eeeeeeee 1180
Nishizuka, Masako .....................000. 1274
INDWial, MIKO sororseansnshernornonepenove Meni EEEE A oleh 1224
IN@WEVNEL, SIMESTOM oo550c000e0ccs000000000000 1268
INOdasIKOUICHIBN A xacey-caaann senna ch Nees a 1190
INO day OSTOMY -rnocnvosictererna Nh tt 1179
Noguchi, Motoko ....................0.0005 1172
Noguchi, Munenori .................. 1218, 1219
INOhAFAMKECIKO RS, ncrrovsroreversnorarcisoryotort eR ae 1247
ING@Yi SUMIN ATC. aeiesicieersoccnsncteeoene en 1201
IN@ypie, SUC Sacenoronosusecesbovospaueus 1299
Nomaguchi, Takashi .....................5. 1214
Nomoto, Kyosuke .....................000 1223
INOMUTAPAKKAT a Reka arscarcovsrorronnci denote 1178
INOMUFA; WON sno. vevereyeresepescrevors ABA SH AE atate 1157
INOMUTARIS CMP srercuarcsdeorctonnccenancvarseasssormel Mae Perera 1152
Nomura, Tsuyoshi ......................055 1268
Nonomura, Yoshiaki ....................... 1146
Nonoyama, Kiyoshi ......................-. 1195
INOS CRT AIS TKN 2 spoxchrensvarcrch onsen nchcnce anno ROM Reh ne 1223
INOSER aS uhinO) PAA. Movessaver ote Meee 1273
Noumura, Tetsuo ........ 1254, 1266, 1268, 1286
WNiovzlisis IMMUIO oosgssncccdussoccduocconcae 1265
INumachteXenichiee eee eee eee 1303
Numakunai, Takaharu ............... 1163, 1303
Numata, Osamu ..................... 1151, 1171
O
Obika, Masataka..................... 1196, 1207
ObinatawAtkiko wirseaiacctaeicrs cy 1286
@binatay Masu0eess eee eee 1287
Obinata, Takashi .................... 1149, 1150
(OXGEWES) NOW tran ans Gaeta eee ere near ote tees 1206
Odaralkalanay vysprercrcrnceronres cerca OM nach 1190
@pashiwaksMasayo ereeyaq-cisaenis eee 1169
@ Paw aseEis ieee GN Neh cece cersrc-o Me ira 1278
@ awwialCaZ OMe r ss pti raeievarerin co ORR Le 1144
@pawiabM NZ Ow ves esyerc alan. cacarseeraste eee te 1258
OsawalMokushise axs.c:0.400e ee 1218
OvisomManabul epee cidade eee 1183
Op UITA, VAWAUITW 5 co gc0nccs00d000csceuecGe00r 1188
OpuraepAtts Omi yet arcsec vrov reverse eae ere 1180
1322
@pruroiChitanul epee eee 1264, 1299, 1303
Ohara TAOS ax. shape css aecisccv oe 1288
Ohashi, Kazuyo ...................-.. 1148, 1149
Ohashi, Shinichi) 2ys..0: cosejrescsic ici ct POR ee 1143
OJoloe, JabTROVOSM scoganec0000s50u00900005c0c 1277
OhizumipWasushil.o4- 02 e ee eee 1148
ONMishigE jie eccmecccicccos eee eee eee 1133
Ohnishi, Fumikazu ......................... 1138
QORMISHI REM Soy Stevosssescdeusvenssscessxsee ee 1237
Ohno; Mada nase c aisscsiersssoviccsn dees 1148
Ohoka,, Tadak az .....:....,.:0ci0c1clerie Ave hel 1145
Ohsuga; engi, eirciecfoesermerensa dee eee 1241
(OMG, IOUS sooacocsgcscc000cc0ccc0a0000 1201
OHSU; Keita, secisjoieesecsieccasinovnt ee Oe 1199
Ohta, Kazumasa ..,.,6.<.s;.c.21:2:.0.01s10 eee 1159
Ohitar MasakOinsiccccucvecaiuiemte cae coe 1183
Ohtas Nadayukd) jc. .scnc-eeee eo eee 1288
Ohta, Yasuhiko ................ 1265, 1266, 1267
Ohtake AIG Skies specced sce Pee oe 1206
Ohtake, Shin-Ichi .................... 1138, 1249
Ohtakisdictsuyaleeeeeeeeeeeeee reer 1141, 1189
Ohtanm@Elidemite-ee eee eee eee ee eee 1239
OhtaniyMasumivee eee eee eee eee ce 1295
Ohtani YURIKO) ses cencucs A eee 1146
Ohitans; Vuk} & iets, scatass verse none eaet 1303
Ohtsuy WohZohy Aseavecccccicti ccna eee 1232
OhtsusiPakashits Aaa sacacccckeo tee ee 1142
OMNITURE, OVO 55 0c0c0cn00000nacccac0c050" 1234
Ohtsuka; Menuyalere-- pee ace eee eee eee 1234
OishiZ NOBORU as ccc eRe 1215
Oishi, Tadashi ........... 1182, 1232, 1233, 1253
1267, 1295, 1296
Oiwas Kazuhiro vescsccipanceeioce osu aeeeeee 1220
Oka Run TO) wes accion eee ee ee 1275
Okay SHUM As sei5 5 secesese. ccs ee 1274
Oka Yoshitaka sso: t.5:0s0:s.ceusvscsssemue eee 1226
Oka Vioshito ms scp seis cscss:sc5,0,000 A CA 1250
OKaGawAts usin jess ogustsojeyecet RI ee 1238
Read ai Din Oi svecisivs dis, dusvosesioysusveak -eustaces ee 1217
Okada Mosk res... 5: jececcacsinces a eee 1211
Okadar My OKO) rege, osepesecesaynessieue AER 1150
Okada, Masukichi ................... 1171, 1188
Okada WOshimora esc scsssssoicssoeeio dee Mle 1235
OkamunraMChisatoy Ain acc.aacccu see eae 1143
OkanoyMoshiy Whales. -1c1c) 2 eeeroree ae 1230
Ok awalWeiko esi kos veveseco csove pases eas 1304
Oki alwashirojsaachiicks scvncs ij. MO OS 1298
Okimaga; SMO oo c.ss siesta reser Mle oe 1197
Okubo, Masaaki ........................00. 1276
Okumura, Elichi ..................... 1161, 1176
Okuno, Makoto ...................... 1144, 1220
@kura, Nobuhiko ..... .-......... eee 1272:
Okuyama, Masahiro ................. 1165, 1166
Okuyama, Ryuhei .....-..-. 25... 1287
@matasSetsuko ee) -- eee eee 1180
Onitake, Kazuo .......... 1171, 1180, 1197, 1198
Ono, Hirotake’ ..... ...45. 66655..6-Reee 1208
Ono; Masatada) 2... ..........:...0..0ee ee Ree eee 1247
Ono; Shoichino: + :,....::..600<48ee eee 1149
Onodera, Kakumasa ......................- 1185
@OnumaysMichiko). ........--- eee 1188
Qofusa,, Ken) sscc08 ili. ccc ce eee 1204
Ookas Hiroshls 22/5) c.c.c0.000 ee 1259
Ookas Vai KO. oss ..6.6..,0:6.6:0e Ree 1293
Ooka-Souda, Sadako................. 1225, 1293
@okata, Kayoko .........20c..0-ceee eo eee 1161
Qota;) Tadachika. .........:.....;....... eee eee 1183
Oni, Midefumiys 2.53 f..:.0000,.000 eee 1186
Osakay TiekO :ii4.0 0). ocjscien bc eee eee 1234
OsanaimiKenziihees eee eee eeeee 1158, 1163, 1207
Osawa; Masaki ........,..:,.0.s0):)::000 ee eee 1206
Oshima, Noriko ................ 1227, 1229, 1274
Ozakis Koichi ye33..3...)..6.05.. 5 eee 1231, 1246
Ozaki, Mamikoos..2%....s0000000 ee 1231, 1246
Ozatos:Kenjitoics.i3 0... eee eee 1209
Ozawa; BLpITO! sce oe. noe Ee 1200
P
Bark Mim Ke sans darsieisjosesenae eee 1254, 1258
Partridge; Juhian'©. <...)...2.2s-eeeeeeee 1230
Periasamy, Muthu... 5.5.2... see 1169
R
Rayborn, Mary BE: .............. 1233
Rosenfeld, Michael G. ..................... 1259
Ryuzaki, Masashi ...................0eeeeee 1187
S
Saegusa; Hironao) .......-. 2.5. -.sesee eee 1269
Saga, Tsuyoshi 2)... .:6....0:00,0,.9bRiodee ene 1257
Sagata; Noriyuki: ....,.........:....jworesise = sree 1278
Saheki, Toshihiko ...................00...5. 1187
Saipa,Hidetoshila..................: een ee See 1192
SAGO MK AOMU ihr. cite na nce naires al Hokie oles 1156
Saigusa, Masayuki .....................0005 1272
Sern] Olay WOUKIO! fee. Wrassesrc nieve Settee oe c SOM 1200
SAITEK ACHE Yotar. urovacorain veh ators Sa ae Rae 1228
SatkiOMOAKI wi ecivnncncaenoedelage ae’ 1177
Saiton DakehikoO)... 06. ..cn cca eens 1235, 1182
SAITOMMASUMON herve ene seeds walee 1176
SaTTOMMO@SAMIU 5.2. ccers ere iece wrote tere eotlalee le whdees 1169
SaltohemMlak ake ccs wiv. HMI 1184
Sakagami, Mayumi.......................5. 1224
Sakaguchi, Morihiko ...................005- 1209
Salat Mie Re mire dian ieannd OAS. «BME 1186
Saka Stowe f sorer coconvoee eo MIAME, RUE 1235
Sakai, Hidetsugu ..................... 1265, 1266
Sais 1810) el re ee ee ree 1146
Sev eiis 1ENSG 0s See ee es 1306
SAIKATEMINOSUIKE? Fey sorescsevereravere rc EARS Ae he 1206
SalkaiWIMakOtol wincsccciveneaecineeee ee 1139, 1263
S KCUIPMIN TAS AKA Moyo poveserarelecsiotesors clbegee Miatehe vols 1225
Sakai, Noriyoshi ...................4- 1174, 1256
Sellar, LE 2 01 0 1284
SADR TDYCOSHNIT dH lovessssseserecereneseccteneatoleted waetbabarels 1179
Salas, WC) 0) Sees seen anne aL ene ne ee 1182
Sakamoto, Naoaki ......................005 1164
Sakamoto, Shinobu ........................ 1258
Salksatat MATIKO Missray.secretorcrcietevererenss ere sleeleeateacle 1223
Saka tay SUSUMU Fak cr.ccssreicscrersvevererare selene ataeccte 1251
Sakuma MAK OtO 6 a ovsjeccrereseyerereecae dees erent 1165
Sakaraio Shots... fk Pcie ces 1141, 1189, 1268
Sakurai, Takayuki ......................... 1172
SakiTals YOUWKO! vivescccccscorescivre ashore fale ealesnals 1214
Salkeurrra Oka UII: ccicneacescss once snctersvotareass MARR vances 1248
S AT OM ARKO 2y.-os¢=rcvosstcnsysvaresavstorevorspate aut. anche 1203
SATO NUIMIKON bovsister cn crcre srcheerssspertel Cae oars 1233
SAOCOMEN KYOKO ih yeresesstcratereven tore Setteas ols 1210
SAS aK UTI Cy. cap cvencncvsrstanctovevoter tae ee cel 1203
SaASAkie MAS (SUISUN. 0...) ey-reses-rsestevor SAI eo oecle 1137
Sas aka INIOTOK OWE occ cyovartvaroveroniolerciarsneterstoredovetelne 1253
Sasalkae SI Opies a srecveyevecccersarniarernenercrarcuasieresiecte’s 1262
Sas alka Mets wl kOy cyssercsevsvoncsor stv oyeicrascvorevereleveronsiete 1152
SAS Aka VAI YUE caniescocsrovsmarsrsronarcranedan ERE ke 1284
Sasayama, Yuichi .................... 1273, 1303
SASS AES jes P Pd bees avaicranersssvsccravcn ee ehedoes 1258
Satake, Shinichiro .......................00. 1268
Sat OPA KI KON i ihrer cvaxorocrcropt ri bee 1274
SALORPAS USING oucrarcuckopovcnsnsronsr Mb eee 1159
Satol G@hikarayy sc rcvsrcisehsxorarcrcenr tomes 1151
SACO MUD AiR patter Power sree vorsrercnvtt ROE 1142
Sato Kats wy i sepcescewsee este othe ot aes 1275
SatOMIN AZM ates eA reeke ero ceessuaus ath hen, BARNE 1194
SatOm MAS AMOLI ii ccsjacc.t i wossessue oid heehee teks 1298
SatonMepumilere cere ncr are aaa cee 1265, 1266
Sato MiIMOKOMAR. ciccceta cess, ete ARO) 1201
SEU), MDT VIR) sonannseonnc0cgndncondnneur 1208
SatomSetsukowriccedcacownwa. Mere NA. teh 1177
Sato ShoOsulkes rey vo saciei i cisscien REE kek 1265
Satoh, Tomomi ................ 1265, 1266, 1268
SatonMlomorsORe becciensaiiecwens theese ute 1245
Satosmioshinobuleeeceeeeee cee ree noeee nen 1276
SCOP GAS USING 22 ss65e cascucceueucaci oe ARMOR Deo cb 1302
SYAIIG)S 2 (0) prota no ee aeRO AL iota tata Am ee 1213
SA LOM OK OURS is csacseace ecsecuu ame hee ae 1160
SATOMI UU CHS foo ssc sesssousas crest occucan APRA, SR 1205
Satoh, Kumiyasu’ .........2.0.:eeee-ssne eee 1305
Satoh, Noriyuki .......... 1140, 1192, 1193, 1300
1301, 1302, 1303
Sato-Maeda, Mika .......................0. 1191
SATOMI, DAISAKUY ...5.cccsepece sore soye ours Ae eee 1183
Swab IMAS AMI. savestessecvensus auevnsh etek SOE 1188
Sawada, Hiroshi ..................... 1135, 1175
Sawadaw Kazulhiko) 5) 5)..4....045se eee eee 1266
Sawada, Norimasa ......................0.. 1142
Sawada, Tomoo ................0 cee eee e ee 1249
Scheuermann, Dietrich W. ........... 1290, 1291
SEIN O MAIN BME osc cedacte nian RRS EE ee ee 1206
Seidou, Masatsugu ....................0000- 1230
SSW Ay WENO) Gaocodoodooqaccds0ccagcca5e 1213
SEK MAK AN ANU ceieccssoieiont errs one 1231, 1233
SekipMatysunOnl oeyemnccmicimerecimeaicce 1183
Sekiguchi, Tatysuhiko ...................... 1241
Seksiyas SKU MO ies seseis tue cores enaynnsseisae ars Met ae vase 1210
SEKOsPA kara ya cssiciotiasSetonescuaenmecae eee 1173
Sen careViltakay Bue. .cccerccurrsc chor Me eeee 1205
SEOMIN AO MMI ess Seccevcrccs suena MOS: 1282
Shibata Michio’ <5:.0-..cecaceieeethe ho ea 1145
Shibuya, Tatsuaki .................... 1225, 1240
Shichida, Yoshinori ............ 1182, 1230, 1232
Shiga akashi acters cuscancint eee aeklae 1183
Shigenaga, Ayako ..................0.0e0ees 1194
Shigenaka, Yoshinobu ..................... 1143
Shikam aye je sacs cree Aa Rds ek 1136
Shimada, Akemi ..................00.000008 1229
Shimada, Hiraku ......... 1143, 1157, 1163, 1164
Shimada, Ichiro ...................... 1246, 1294
ShimadabwMatsuya: a-.accicie ssn she ascend: 1304
Shimasaki, Shunichi........................ 1156
1324
Shimazakikeniilleeeeee sree nee ee eee eens 1244
Shimazaki-pYOuiyigeee- eee eee er eeeeee 1237
Shimizu SEMTOSh .cssccsvcisenswscice ee 1240
ShimizuesisamUleeeeeeee eee eee eee 1238, 1271
Shimizu, Katsuhiko .................. 1158, 1179
Shimizus Nek OW di... ciescrjonennieniota eee 1265
Shimizu, Munetaka =.5....-..5...--eaeeeee 1244
Simizus, PakaltikOws4...<c01ecesiecvcievsvere Se 1167
Shimizu, Takashi............... 1143, 1144, 1220
ShimizwaMlakashin. sce. cee oe eee 1209
Soirmmivan, Walkees it 55 55ccncencccasaaccucaconse 1205
Shimizu, Toshinobu .................. 1241, 1242
Shimizu, Yoshinobu ........................ 1206
ShimodasNobuol..---- eee LeeLee 1139
Shimoday Yasushil.s....--sceeeeooe ween. 1174
Shimohigashi, Miki ........................ 1236
Shimozawa, Atsumi ............ 1204, 1248, 1249
1288, 1290
Shimozawa, Tateo ............. 1181, 1241, 1242
Shimurayiakumileee eee eee ee eeern ree 1218
Shingyoji, Chikako ......................00- 1219
Shinkai, Maas ..c.jscyeyercnsonensvereicis eee te 1259
Shinkawa, Hideaki ......................... 1211
ShinodalwictsunoO peepee eee eee Eee Eee eee 1152
Shinozawa, Takao ....... 1185, 1187, 1230, 1300
Shinozuka, Kazumasa ...................... 1216
Shiojiri, Nobuyoshi .................. 1184, 1208
Shiokawa, Koichiro ........................ 1156
Shioya, Masakatsu ...................0.008- 1265
ShiozakiMSyuich), .;.yc,ececac cinerea 1187
Shirai, HILO KO oy. jorysiceecssuncdncine chee aoe 1158
ShiraiaManabuyyasas- cece eee Eee eee 1260
Shirai, ToShiOise sss ev.csaunsindcnaivas Peck 1183
Shiraishiaisatoshilea-eaceteceeeeeeneneer WH.
Shirasawa, Mikio .....................0000 1196
Shimasawas YaSUKO) o....:cc00.0 eyes rescind eee 1282
ShitayamasexOshihisa) jasc. cee eee eee 1300
Shirayoshi, Yasuaki .....................00- 1172
SHOWA VOIO! [550s cs: 0syesovseueere dO ee 1179
Shishikura, Fumio ................... 1138, 1149
SHUM Gaia ri snes siiscdeieresdiecoussie MA Soe 1220
Sieber-Blum, Maya ...................0.005 1208
Singtripop, Tippawan ....................65 1258
SOLA AMO MOM Uy csaeseicssesorere neers ATO 1216
SOMA TUM Dea le tes tes ciessuavorterrorntyetnt teks Peele 1189
SOME MVAMIK OF hy ehecevctevsrsrocorseioceveioers SA 1187
Sonobe, Haruyuki ....................00008 1270
Strausfeld, Nicholas J. .................005. 1225
Sudzuka\Minomil ¢ 220-005... - eee eee
Sueoka Merumi\........:::0.:.0 - nee eee
Suganuma, Yoshiko........0....5. =e
Sugase, Yasuko
Sugawara, Miho
Supi, Haruo’ 33.5665 6. eae le
Sugihara, Shuji
Sugimori, Shin
Sugimoto, Masazumi .......................
Sugino, Hiromu ......................
Sugiyama, Kazuo
Sugiyama, Takanor ........................
Sugiyama, Tsutomu ........................
Suhama, Mikio
Sumida, Masayuki
Sutoh, Kazuo
Suyemitsu, Takashi
Suzuki, Akinori
Suzuki, Atsushi
Suzuki, Atsushi
Suzuki, Haruhiko
Suzuki, Hideyuki
Suzuki, Hirobumi
Suzuki, Hiromi
Suzuki, Jun
Suzuki, Kazuo
Suzuki, Makoto ....,...<...0:...;00:0ce eee
Suzuki, NOBUO: 3... ./.,..<.-,<:6.-;5::,.000e Se
Suzuki, Norio
Suzuki, NOvIyO ..0..)....< ccc 2000. ke eee
Suzuki, Sachiko ....4.0.....6:6..0:+.yi.10 2 ae
Suzuki, Shintaro
Suzuki; Taketo: s..ccs.o.csceis0s, eee
Suzuki, Takuro
SUZUKI, Tatsuo: ..o.0.<.c:sece;0j0101000y0, 02 ee
Suzuki, Tomohiko
Suzuki, Yoshiaki ...........:.:.0:..0... 2g eee
Tabata, Hiroshi
Tachibana, Kazunori .................. eae
Tada,, TOMOKO. .....jc..s..e.0:ee0e eee
Tagawa, Kunifumi
Tagawa, Masatomo
Taguchi, Tomohiko
Taguchi, Yasuko: «.5.:..6ssyeeriscsosio OR
Tajika, Ken-Ichi
1148
1269
1196
1245
1138
1301
Majimas!Genaiehth oats -..ce. ane hisiee leo eleioe 1136
MakabatakeTKUO) ........0:..00.. 28 eetiee onstine 1228
PRANK aCe MD UKG aha ein cies esses assiejeieienelsleie Sele 1220
Takagi, Takashi .......... 1140, 1174, 1248, 1301
BAKA IR GLOTUN: Esti. :s)sj0.ocn,einyeie seis blqametaettete: 1294
AEA eAPTEVOSIIOMII ib o.66.5. ie oi 6 nie sien s « aes 1278
Makalnamal, HIG «5. ce cceceviee sisetoeloe « 1234
Makalara= IAT y..:0..:.ceriaeeoyese ceed satel oe 1161
Takahahshi, Fumie ................... 1145, 1146
Takahashi, Hiroya ................... 1197, 1257
Takahashi, Keiichi ....................00.0- 1219
Takahashi, Kyoh-Ichi ...................... 1235
Takahashi, Mihoko ....................005. 1212
Makahashis Mika: ..........50 <<... < sufaentemenia ete 1256
Takahashi, Nobuko ........................ 1172
Takahashi, Noriyuki ................. 1261, 1262
Makahashiy SOW o\.c.cec. ces e eee Dees 1247
Takahashi, Sumio .............. 1258, 1259, 1260
Makahashis Susumu -...............ss5008-- 1139
Takahashi, Tadao ........ 1276, 1278, 1279, 1280
Takahashi, Tadashi C. ..................... 1154
Makahashw, Toshio)... 5.22... .s0c.s oe 1223
TEV@INE OV QU er eee 1168
Takahata, Masakazu ....................... 1224
Paka SAKU 224s eh 2 «eee esses a cbo,o Seay 1168
Takai, Michikatsu ......................0005 1176
Takakuwa, Michiyo ..................-...-. 1196
Takamune, Kazufumi ................ 1198, 1199
Takano-Ohmuro, Hiromi .................. 1147
Makasumy lide Oma tir a. perce eee wee ote takes 1139
Takasugi, Noboru ........ 1265, 1267, 1268, 1273
MakatasMazukOy sd . coc cis eee cess me eee 1163
MakataeWenZOns cs e26 6b eines euscve aise PF 3GS Re cs 1155
MakatasiKumniakd ©22.............sgrisetiok. sien vack 1250
Takatsuki, Yoshiko ......................5. 1243
Makayas Susumu. 5... 22... -yfeesrecesssneee 1279
Takeda, Masatsune ........................ 1305
Makedas Shin=Ichil \............. sane eee 1295
makedatSoichiiey: § s<s5 0. cuceece BESO alee 1165
MAK VOSHION KG... salaateh.<ehaeee Me doeet! 1273
Makemasa; Tohm i.......5 0.00.0. .0 tise. ct 1212
Takemoto, Kazuhisa ....................... 1175
Takeshima, Kazuhito ...................... 1154
MakesucttSachikOes 6... eccccksee so oe 1155
MakeuchisHino-akal 45 5....0.... she tee ae 1244
MakeuchieSakae. sc . o.8 occ esc ne cere 1260, 1285
MakeuchisShigeow. can. sede ote 1184
MakeuchiyMakcyjiees-ceeee. -eeeereee 1208, 1213
Takeya, Akira
Takikawa, Shin-ichiro ......................
Mhakizawa> Satoshi joc as «0 soeloupeererr as» cess
Tamate, Hidetoshi B.
Tamotsu, Satoshi
Tamura, Koji
Mamuras SachiKOy. cae oss 5-1.5 sehen senk «cots
Tamura, Toshihiro
Tamura, Toshiki
Tanabe, Kazuyuki
Tanabe, Ryota
Tanaka, Akira
Tanaka, Haruo
Tanaka, Hideaki
Tanaka, Hideki
Tanaka, Kosuke
Manaka wKUMIOe:. cs sce. 4 eee
whanakaw Masaaki. cc: seeps ope:
Tanaka, Midori
Tanaka, Minoru
Tanaka, Mitsuo
WWanakarRy Ol ns caccs.: dadedaaad este
Tanaka, Shigeyasu
Tanaka, Shingo
Manakatl, Shojity. 206. cee se soos cates
Tanaka, Takashi
Tanaka, Tetsuya
Mian dai Ofleee Peis coec eis a EEE cee
Taneda, Yasuho
Tani, Tomomi
Tanigawa, Hisashi
Manimura,Teiichi ...........idacdadadd.-assee
MashirowhideOrenw eee eee eee eee
Tatematsu, Ritsuko
Tatunami, Shinobu .........................
MawarawAkihikoin gg. .006.50 teeta oda
Tazaki, Kenro
Tazawa, Eigoro
Tegner, Jesper
Terakita, Akihisa
Teramura, Wataru
Terano, Yasuhiro
Terasaki, Asako
Teshirogi, Wataru ....................
Timmermans, Jean-Pierre ............
Titani, Koiti
Tochinai, Shin
1217,
1138,
1139, 1255,
1262,
1184,
1290,
1222
1249
1239
1222
1133
1192
1261
1263
1227
1226
1148
1185
1291
1135
1326
Toh YoOshihitoOpest ee eee eee eee 1236, 1237
Tokudas'Gakw o.ncs2c20 eee ee 1281
Tokumoto, Toshinobu ..................... 1162
Tokunaga, Fumio .............. 1182, 1231, 1246
Tokusumi, Yumiko ........................ 1278
Tominaga, Hideyuki ....................... 1292
Tominaga, Takashi......................... 1218
Tominaga, Yoshiya ...................-.-.. 1236
WOMINO -SHITOY hotest ee 1141
Momiokas Kenyie---- eee eee 1238, 1295
Tomioka YuKie> «..:..6c..cle ee 1279
Tomitas Hideo... eee 1209, 1214
Tomonaga, Susumu ........................ 1249
Tonomura, Yasuko .....................0- 1211
Monosakia Neichit-.- eee eee eee 1245
Moratanis| Satoshi \s..cce eects eee oe 1176
Toriyama, Masaru ..................-2-00-+ 1176
Toriyama, Mayumi ....................-.--- 1141
Tosujis Hiroakiey.aceeee. eee ee 1145
TOIT, SWOT 5 5000000000d00c0000G00000C 1146
Toyoda, Fumiyo ................--.--0--+-s 1255
Toyohara, Haruhiko ....................-.. 1209
Toyohara, Jun isc cecrcen 1229
HoyoizumibRy uel eee eee eee eee eee 1184
Moyoshimayp Oko) a. ee eee eee 1143
Tozakil Shizuka ic neem ee 1247
Truman, Jamesews s.005..62ne7.c eeeeoeee 1271
‘suchiya,. Takeshiltyycicctirn rare eee eee 1302
Msuchiyas TEiZO Wee cesses ene ee 1215
Psujie Lakashive..pr.cesnn ent eeee aoe 1184
Tsujimura, Hidenobu ...................... 1271
Tsukahara; JUuNZo..4:ennncc eee ee 1159
Tsukahara, Masatoshi ....................-- 1151
Tsukahara, Yasuo .................... 1228, 1233
Msukuda) Aaroko ...cccccic ee ee 1217
TSW) I, NREVAUIWKO) coococccoccnbd0soo0000000 1281
Tsuruhara, Takashi ........................ 1277
Tsuruta, Tatsuhiko ......................... 1167
MSUSUE="NIOCOO «hora. arerensts ls toterarasoromele eo 1135
Tsutsui, Kazuyoshi .................0ee ee eee 1266
aisutsumisladaakil cnn eee 1284
TUTON NAVIET Phe Siete eennce win eee ree 1303
U
Wehiday Katsuhisa s...cehiee exter se eens 1303
Uchiyama, Hideho ................... 1155, 1156
Uchiyama, Hiroyuki ....................... 1206
Uchiyama, Hiroyuki ....................... 1234
Uchiyama, Minoru .......................-. 1263
WedarBrnA Gen. weariness ee 1176
Weda Hiroshi... 285)... ee 1170, 1244
Weda, Masatsugu <5... eee 1251
Veda, Ry ..102.6:00c einen nee eee 1233
Weda; Shui )..:..:0.:.0:..:..0. eee 1239
Wehara, Hiroko 2.00.00. \o0nn neh eee 1244
Ueharas Seiji: 22. wen). ene ee 1252
Ueki, Tatsuya 0:00.) 1193
Wemura; Haruko .........7.05 eee 1260, 1261
Weno, Naoto: oilec icc ee 1156
Weshimas Rel. eee 1283, 1300, 1303
Ukawa, Ken-ichi «...... ...:.-..:.:...0%1 eee 1273
Umebachi, Yoshishige ..................... 1134
Umeda; Takashi: «............. eee 1220
Umino, Osamu -..........0./..:.0 0 eee 1236
Urano; Akihisal ..c:.c:.:.:000:..0 ee 1267
Wrase, KOKO ~. ....:01s ser se 1194
Wsami, Mihoko .....\. <0. «eee 1138
Ushiyama, Akira ...................0.00000- 1197
Usui; AKIKO 2h. cicccnrscron ote eae 1243
Usui, Kazuya.) ooo. ect cintee ee 1189
Usui, Noriko d..0.:e.:e.n%00 ee 1180
UWto, Noribiko «.-.-.0.:0.0.% 000k ee ee 1176
Utsugi, Kazuo 2... cops 1297
Wyama, Taro ..s:0.:.).1.)-n ences eee 1250, 1251
W
Wada, Hiroshi ................. 1301, 1302, 1303
Wada, Masaru® . ..:0 coco nrete Oe 1255
Wada Shigeo’... chee eee 1144
Wakabayashi, Katsumi ......... 1183, 1254, 1261
Wakabayashi, Shuichi ................ 1265, 1266
Wakahama, Ken-Ichi ...................... 1188
Wakahara, Masami .................. 1154, 1264
Wakamatsu, Yuko ....05...5<. dene 1209
Washio, Hiroshi. .....:........0. ose 1242
Washitani-Nemoto, Setsuko ................ 1177
Watabe, Shoji ....ccc.s omen eee 1138
Watanabe, Akihiko! .......:...%ees eens 1201
Watanabe), Kazuo «..)..:...05 otaveentnoenenene 1200
Watanabe; Ken’ 2.0... 0.0005... e eee 1277
Watanabe; Kenji ............... Se eaeweee 1178
Watanabe, Kenji <......... 00.5 seen 1186
Watanabe, Satoshi ..................0.0000s 1148
Watanabe, Shinichi .....................0.5 1237
Watanabe, Tomohiro
Widtanabe. YOKO) sccccscecu00t cons vecieehes
Watanabe, Yoshio
Watanabe. YUICHI! 2... .0.00ceese sees cece ces
Wataya, Yusuke
Weibrecht, Josette M.
Wuchiyama, Junko......................05.
Yabuuchi, Masafumi .......................
WiaSieMYOSHIMASA) ..... 260 5eee seine as cena
Yago, Nagasumi
Yahata, Kensuke
Yamaai, Tomoichiro
Yamada, Akira
Yamada, Atsushi
Yamada, Kazumi
iat aAC AMOI ee cin vais oie cuneieninvacinve ec ee aces
Yamada, Osamu
Yamagami, Kenjiro
Yamagishi, Hiroshi
Yamaguchi, Kelichiro ......................
Nama cuchibakeO syne acese. sees cee:
Yamaguchi, Tsuneo ..................
Yamahama, Yumi
Yamakawa, Yoshio
Yamamoto, Daisuke
Yamamoto, Hiroaki........................
Yamamoto, Kazutoshi
Yamamoto, Kiyotaka
Yamamoto, Mari
Yamamoto, Masamichi
Yamamoto, Takashi
Yamamoto, Toshiharu
Yamamoto, Yoshimi .......................
Yamanouchi, Korehito .....................
Yamaoka, Ikuo
Yamasaki, Kenji
Yamashiki, Naoko
Yamashiro, Hideyuki
Yamashita, Kaoru
Yamashita, Masakane..........
Yamashita, Satoru
Yamashita, Shigeki
Yamasu, Kyo
Yamasu, Terufumi
Yamauchi, Kohei
1260
1202
1229
1215
1222
1287
1176
1164
1139
1255
1246
1237
1281
1327
1244, 1256, 1257
Miamaura, ELISASII sac -sys)suesssecsrevoe SN oe 1277
Namazakis Hinoko)aveeeeeneeeeeeae sneer 1134
Meamazakin Wen as cticechee ates 1143
Yamazaki, Tadashi......................04. 1196
Yamazato, Kiyoshi ......................... 1292
Yanagimachi, Ryuzo ......................- 1180
Yanagisawa, Tadashi ....................... 1263
anal NO DUA Mercier rece eet te ee 1287
RYAN OMINeM=1Clih ee kas socusntesiens RAT hee 1141
VWAO, MAGI copacosoncooogsonnneunsoaes 1240
BVA OR SYA OM rience icrsvessroxcistseceoare od PRE ct 1169
BY¥ashimmas PY O1 Chit ees, icesicusrevss vorccecrson eee 1276
RYAN se VAUIT es eersjctcy (elec natininlee mente altos 1233
Nasu cietsukoy tere acsier ear acne 1194
DASUCIRS Ada Ota ce aecisae acl asec eter. 1194
Yasumasu, Ikuo ......... 1137, 1143, 1165, 1166
1167, 1168
Wasumasus shigeki senses ase eer 1157
VASUO, IaNOKOSM 5occuccco0ocsc00s00c0undoe 1193
Yasutomi, Masumi ................... 1188, 1285
Yau KGNPoWiat on ach cecsamesincenee seen: see 1234
Yazaki, Kazumori.............. 1143, 1145, 1146
Wazawar SMM eee ei oc ciccgncyewme vale ee 1139, 1170
Wazawab Mohnu® ccs... <ccaenaswe es sevcse ceneeae 1222
Yazawa YOICh sec. cccesesSeecssen. 1215, 1216
SOKO AIM, IEWTNVO® 5 osceccnccacaeacancunocans 1242
Yokosawa, Hideyoshi .......... 1163, 1175, 1176
WokosawaniNOnkoOnmnnsenadeee eee eee 1228
WokosukamMakotomeeeeenc eee tere en neee 1265
Yokotal Kaztimant oi ces cccccdeavensebecees 1244
Yokotarvukior ss cc cascanciw snes 1178, 1285
YokouchiM Yuji tee sacess saree sees 1200, 1201
Wokoyassachihikoweren-eeeeeeee eee eee 1209
NOROWaN, MRO 2 scaccc0000000000000000006 1269
WOkoyalnasi nO sees eee here eee caer ren reer 1192
Yokoyama, Sigeyuki ....................... 1150
Yokoyama, Takehiko ...................... 1210
YORMOR, SVGOMONU cooncvascesncossus000dse 1204
NOREGA, IMTEUIS, coccoccoconoossaebesuoonse 1163
NONEIMS ayn e ery Ae a aonnens mes kee: 1201
Yonezawa, Yumiko ........................ 1286
WoroisakavASakO) joccccccsn sca ceses sees 1199
Yoshida, Akihiro .................... 1189, 1227
MoshidawWelichiter sae eee seceee eee an: 1202
NORM, UQWOKO so0c00cccca0cn009000000000¢ 1300
hWoshidasVanabumeerneeeeerneeeeeaaeeee: 1206
NoshidaMMiasSaOM. ates h cee ba eee ee bareane 1232
1328
Yoshida, Masayuki .......................-% 1222
YOshih ara HAZ UO lic oscccrssacrcoce ee eee 1252
Yoshihara, Masayoshi...................... 1264
Yoshihara, Yumiko ........................ 1149
Yoshikawa, Tomoko .....................+- 1233
Yoshikuni, Michiyasu ...................... 1162
Yoshimura, Kenjiro ........................ 1219
Woshizaki,, NORM. 6.0.cncaen eee ee eee 1174
Yoshizato, Katsutoshi ................ 1151, 1204
Yoshizawa, Tohru ....... 1182, 1230, 1232, 1233
MWosizawa, IAIdeKI ..::..c.c.c.comnins eee 1303
iuasal Hajime di. .jascm.cicasciecdee heer 1301
SYA TA’ Mam OR a. Aciereve. vere coresenciencron CO RE 1152
Z
Haow XaUemell feat a Ae scvocoescn AOL 1236
AGO TAT CES Cale eyes a theo wonieioen ane eee 1178
ACKNOWLEDGMENTS
1329
The editors express their gratitude to the following reviewers, who evaluated papers for
ZOOLOGICAL SCIENCE Volume 9.
Abe, Hiroshi
Abe, Shinichi
Ae, Shigeru
Ai, Naohiro
Aida, Katumi
Aoki, Junichi
Aoki, Kiyoshi
Arai, Yasumasa
Arikawa, Kentaro
Asashima, Makoto
Ashida, Masaaki
Baba, Keiji
Chiba, Motoe
Chiba, Yoshihiko
Eguchi, Eisuke
Endo, Katsuhiko
Fujii, Ryozo
Fujishima, Masahiro
Fukuda, Hiroyuki
Fukushi, Tukasa
Furuta, Emiko
Gotoh, Toshio
Hara, Reiko
Hara, Tomiyuki
Harada, Eiji
Harada, Tetsuo
Harumi, Tatsuo
Hattori, Atsuhiko
Hayashi, Shinji
Hayashida, Kazuo
Hikida, Tutomu
Hirano, Tetsuya
Hirata, Shuji
Hisada, Mitsuhiko
Honma, Yoshiharu
Hoshi, Motonori
Hyodo, Susumu
Ide, Hiroyuki
Iga, Tetsuro
Iguchi, Taisen
Imajima, Minoru
Inoue, Shojirou
Inoue, Yasuo
Ishibashi, Takaaki
Ishida, Sachiko
Ishii, Naokata
Ishi, Saburo
Ishii, Susumu
Ishikawa, Hajime
Ishizaki, Hironori
Tuchi, Ichiro
Iwahori, Nobuharu
Iwamatsu, Takashi
Iwasaki, Shinichi
Iwasawa, Hisaaki
Kagawa, Hiroaki
Kamishima, Yoshihisa
Kaneko, Toyoji
Katakura, Yasutoshi
Kato, Yukio
Kawakatu, Masaharu
Kawamura, Kazuo
Kawamura, Kosuke
Kawanabe, Hiroya
Kawashima, Seiichiro
Kikuchi, Yoshiaki
Kikuyama, Sakae
Kineya, Toshiaki
Kinoshita, Tsutomu
Kishida, Yoshikazu
Kishimoto, Takeo
Kitou, Yuji
Kobayashi, Hideshi
Without their assistance the journal could not function.
1330
Kobayashi, Jun
Kobayashi, Kan
Kobayashi, Makoto
Kobayashi, Tohru
Kobayashi, Yasuo
Koizumi, Sadaaki
Kosaka, Toshikazu
Kosuda, Kazuhiko
Kubokawa, Kaoru
Kuchiiwa, Satoshi
Kuwasawa, Kiyoaki
Machida, Masaaki
Machida, Takeo
Maeki, Takamichi
Matsuda, Kohei
Matsuda, Motoko
Matsumoto, Akira
Matsumura, Akiyoshi
Matsui, Masafumi
Matsuoka, Norimasa
Mawatari, Shunsuke
Michibata, Hitoshi
Michinomae, Masanao
Miura, Tomoyuki
Mogami, Yoshihiro
Mohri, Hideo
Mori, Takao
Morimoto, Katsura
Morisawa, Masaaki
Moritani, Tsuneo
Moriwaki, Kazuo
Muneoka, Yojiro
Murakami, Akio
Nagahama, Yoshitaka
Nagao, Takashi
Naito, Nobuko
Nakamura, Masahisa
Nakamura, Shogo
Nakano, Eizo
Nakatani, Isamu
Nakayama, Shin
Nambu, Hisao
Niida, Akiyoshi
Nishida, Hiroki
Nishikawa, Teruaki
Noguchi, Motoko
Nojima, Tetsu
Noumura, Tetsuo
Nozaki, Masumi
Numakunai, Takaharu
Nunomura, Noboru
Nunomura, Wataru
Obara, Yoshiaki
Obinata, Takashi
Ogasawara, Tsuyoshi
Ogawa, Mizuho
Oguro, Chitaru
Ohta, Hisashi
Ohta, Yasuhiko
Ohtsu Kohzoh
Oishi, Tadashi
Oka, Yoshitaka
Okada, Masukichi
Onishi, Exji
Onitake, Kazuo
Ono, Hirotugu
Ooami, Kazunori
Oota, Yoshihiko
Ootaki, Tetsuya
Osanai, Kenji
Sado, Giichi
Saiga, Hidetoshi
Saigusa, Masayuki
Sakai, Hidetsugu
Sakai, Masaki
Sakamoto, Tatsuya
Sakurai, Shou
Sasagawa, Hiromi
Sasagawa, Mituhiro
Sasayama, Yuichi
Sato, Katsuyuki
Sato, Noriyuki
Sato, Takanori
Sekiguchi, Koichi
Seto, Takeshi
Shikama, Keiji
Shimada, Hiraku
Shimada, Ichiro
Shimazu, Takeshi
Shimozawa, Tateo
Shingyouji, Chikako
Shinozawa, Takao
Shiokawa, Koichiro
Shirai, Hiroko
Shiraishi, Akio
Shirayama, Yoshihisa
Shirotani, Youko
Soneoka, Terumi
Sonobe, Haruyuki
Sueoka, Terumi
Sugawara, Takashi
Sugi, Haruo
Sugiyama, Kazuo
Suzuki, Kunio
Suzuki, Norio
Suzuki, Shintaro
Suzuki, Tatsuo
Tagawa, Masatomo
Tajika, Ken-ichi
Takahashi, Hiroya
Takahashi, Mihoko
Takahashi, Nobuaki
Takahashi, Sumio
Takahashi, Susumu
Takasugi, Noboru
Takeda, Masatune
Taketomi, Youko
Takeuchi, Kakuji
Tanabe, Noriko
Tanabe, Yuichi
Tanaka, Shigeyasu
Tanimura, Teiichi
Tazaki, Youko
Terakado, Kiyosi
Tojo, Sumio
Tomino, Shiro
Tomioka, Kenji
Tomita, Hideo
Tsuneki, Kazuhiko
Tsutsui, Kazuyoshi
1331
Uchiyama, Minoru
Ueda, Hiroshi
Ueno, Shunichi
Ueno, Teruya
Umezawa, Shun-ichi
Urano, Akihisa
Uwa, Hiroshi
Wada, Masaru
Wakabayashi, Katsumi
Washio, Hiroshi
Watanabe, Yuichi
Yamada, Mayumi
Yamagami, Kenjiro
Yamaguchi, Kelichirou
Yamaguchi, Masaaki
Yamaguchi, Toshio
Yamaguchi, Tsuneo
Yamamoto, Kazutoshi
Yamamoto, Masamichi
Yamanouchi, Hiroshi
Yamanouchi, Korehito
Yamasaki, Kenji
Yamashita, Satoru
Yamasu, Terufumi
Yamuchi, Kohei
Yamazaki, Hiroko
Yano, Koji
Yasugi, Sadao
Yasumasu, Ikuo
Yata, Osamu
Yokosawa, Hideyoshi
Yonezawa, Satoshi
Yoshida, Akihiro
Yoshihara, Masayoshi
Yoshikuni, Michiyasu
Yoshizato, Katsutoshi
Yui, Ryogo
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ZOOLOGICAL SCIENCE
VOLUME 9 NUMBER 6 DECEMBER 1992
CONTENTS
REVIEWS
Scharrer, B.: Recent progress in comparative neuroimmunology.................0-2-eeeeeeeeeee 1097
Gorodilov, Y. N.: Rhythmic processes in lower vertebrate embryogenesis and their role for
developmentalicontrol 2 3.0... cg cRPs fei ooo nig: seine ed - ose 1101
Suzuki, K. and D. G. Furth: What is a classification? A case study in insect systematics:
Potential confusion before’order . 2.2.2.5... sen++--- 2 5042 )2 2) oe eee eee 1113
PROCEEDINGS OF THE 63RD ANNUAL MEETING OF THE ZOOLOGICAL SOCIETY OF JAPAN
Abstracts of papers presented at the 63rd Annual Meeting of the Zoological Society of Japan ....1127
Abstracts of papers read by the Zoological Society Prize winners
Takeuchi, T.: Gene expression involved in melanocyte differentiation in the mouse .... 1129
Yamagami, K.: Studies on the hatching enzyme and its substrate, egg envelope, of
Oryzias latipes: cies ccacenawnratees soameeiness oven okie 600 cse 00ers 1131
Biochemistry ................ paladin sideaisia Belo? Mwere ould «eee aielote eee eee 1133
Developmental Biology acc. .cccscedingeniens dee teedinn coteses onde sceses en 1154
GENECS ween ese Reecenhe wena erisne ecemuieg ete slostosuren se oor ne See gee aaa 1210
PhYSIOIOSY wee saek eee ode tine ood ods ectnee Sees eo semeee de wie lt “ele eee or 1215
En@OcrinOlogy wesis sitesi s cusralocie ten eag edwiois ose oiese cee hie olereisier ee absietsleecle 1254
Cell Biology and Morphology 2 os.c..c0sc0 05 eb eciiee estes wciee cieiee eins see 010s ee 1275
Behavior Biology and! Ecology... 2:00. 000060005 cees soe ceee seme. eo eee eee 1292
Taxonomy and SystematicS: 2.0... 0.20. ceene tents e eee eee tok senile secede 1297
AMMOUNCEMENES! ook. k eddies ecwpee eae edn e oe eleleais sie calor ene ore ode eee gies Siehiere oleate 1307
Acknowledgments 2 ..c0d.e cca wete sociesteaned ees Mem oe slobinnae gs omieieleledid 5o ee lOe 1308
Author inde oicsses dan consndmcnda ten enone ee eRe 1 nee cimenaemtak ne Geto se coe eee ee rr 1309
Contents of ZOOLOGICAL SCIENGE; Voll 9; Nose 126) 22 yaiica. ee ee eee i
INDEXED IN: Issued on December 15
Current Contents/LS and AB & ES, Printed by Daigaku Letterpress Co., Ltd.,
Science Citation Index, Hiroshima, Japan
ISI Online Database,
CABS Database, INFOBIB
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